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Excitation energy transfer between the photochemically active protein complexes is key for photosynthetic processes. Phototrophic organisms like cyanobacteria experience subtle changes in irradiance under natural conditions. Such changes need adjustments to the excitation energy transfer between the photosystems for sustainable growth. Spectroscopic assessments on purified photosystems usually fail to capture these subtle changes. In this study, we examined whole cells from two model cyanobacteria, Synechocystis sp. PCC 6803 and Synechococcus elongatus UTEX 2973, grown under high and low light conditions to decode the high light tolerance of the latter. This allowed us to study photosynthetic machinery in the native state and in this work we particularly focused on the excitation energy transfer within PSII and PSI manifold. Understanding the high-light tolerance mechanism is imperative as it can help design strategies for increasing the light tolerance of cyanobacteria used for carbon neutral bioproduction. Our observations suggest that Synechococcus 2973 employs an uncommon photoprotection strategy, and the absence of hydroxy-echinenone pigment in this strain opens the possibility of an orange carotenoid protein homolog utilizing zeaxanthin as a scavenger of reactive oxygen species to provide photoprotection. Furthermore, the adjustments to the high-light adaptation mechanism involve downregulating the phycobilisome antenna in Synechococcus 2973, but not in Synechocystis 6803. Additionally, the stoichiometric changes to PSII/PSI are more tightly regulated in Synechococcus 2973., Competing Interests: Declarations. Competing interest: H.B.P. reports financial support was provided by Washington University in St Louis. D.M.N. acknowledges the Center for Solar Energy and Energy Storage at McKelvey School of Engineering at Washington University in St Louis for financial support., (© 2024. The Author(s), under exclusive licence to Springer Nature B.V.)

The main protease (M pro ) is an attractive drug target for inhibiting the coronavirus. Lots of research has focused on the static viewpoint of the M pro , such as the X-ray crystal structure, inhibitors design, and the transition between monomer and dimer. However, the attention to the dynamical features of M pro is limited, which is essential for a deeper determination of the properties of the target protein. In our research, we constructed three single-tryptophan mutants (W31IN, W207IN, and W218IN) of M pro from SARS-CoV-2 and SARS-CoV to monitor the motion of M pro at the nano-second timescale using the time-resolved fluorescence assay. We found that the temperature-dependent Stokes shift results show various behaviors among the three single-tryptophan mutants: the microenvironment around the Trp207 residue is more temperature-sensitive compared to that in residues Trp31 and Trp218. The molecular dynamic simulation results further support that M pro SARS is more flexible than that of M pro SARS2 . This difference is directly related to the distinct perturbations of residues Phe185 to Gln192, a loop that connects domain II and domain III. For the first time, we were able to reveal the different motions between M pro SARS2 and M pro SARS , although the static structures of these two are not distinguished. The differences in dynamics would be an essential step towards understanding the evolving trend of coronavirus, providing a comprehensive view of the properties of M pro , and offering perspectives for designing inhibitors for further research., Competing Interests: Declaration of competing interest The authors declare that they have no competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024. Published by Elsevier B.V.)

Objectives: One of the widely used optical biopsy methods for monitoring cellular and tissue metabolism is time-resolved fluorescence. The use of this method in optical liver biopsy has a high potential for studying the shift in energy-type production from oxidative phosphorylation to glycolysis and changes in the antioxidant defense of malignant cells. On the other hand, machine learning methods have proven to be an excellent solution to classification problems in medical practice, including biomedical optics. We aim to combine time-resolved fluorescence measurements and machine learning to automate the division of liver parenchyma and tumors (primary malignant, metastases and benign tumors) into classes., Materials and Methods: An optical biopsy was performed using a developed setup with a fine-needle optical probe in clinical conditions under ultrasound control. Fluorescence decays were recorded in a conditionally healthy liver and lesions during percutaneous needle biopsy. The labeled data set was created on the basis of the recorded fluorescence results and the histopathological classification of the biopsies obtained. Several machine learning methods were trained using different separation strategies of the training test set, and their respective accuracy was compared., Results: Our results show that each of the tumor types had its own characteristic metabolic shifts recorded by the time-resolved fluorescence spectroscopy. The application of machine learning demonstrates a reliable separation of the liver and all tumor types into cancer and noncancer classes with sensitivity, specificity and corresponding accuracy greater than 0.91, 0.79 and 0.90, using the random forest method. We also show that our method is capable of giving a preliminary diagnosis of the type of liver tumor (primary malignant, metastases and benign tumors) with a sensitivity, specificity and accuracy of at least 0.80, 0.95 and 0.90., Conclusions: These promising results highlight its potential as a key tool in the future development of diagnostic and therapeutic strategies for liver cancers. Lasers Surg. Med. 00:00-00, 2024. 2024 Wiley Periodicals LLC., (© 2024 The Author(s). Lasers in Surgery and Medicine published by Wiley Periodicals LLC.)

Photosystem I in most organisms contains long-wavelength or "Red" chlorophylls (Chls) absorbing light beyond 700 nm. At cryogenic temperatures, the Red Chls become quasi-traps for excitations as uphill energy transfer is blocked. One pathway for de-excitation of the Red Chls is via transfer to the oxidized RC (P700 + ), which has broad absorption in the near-infrared region. This study investigates the excitation dynamics of Red Chls in Photosystem I from the cyanobacterium Thermostichus vulcanus at cryogenic temperatures (77 K) and examines the role of the oxidized RC in modulating their fluorescence kinetics. Using time-resolved fluorescence spectroscopy, the kinetics of Red Chls were recorded for samples with open (neutral P700) and closed (P700 + ) RCs. We found that emission lifetimes in the range of 710-720 nm remained unaffected by the RC state, while more red-shifted emissions (>730 nm) decayed significantly faster when the RC was closed. A kinetic model describing the quenching by the oxidized RC was constructed based on simultaneous fitting to the recorded fluorescence emission in Photosystem I with open and closed RCs. The analysis resolved multiple Red Chl forms and variable quenching efficiencies correlated with their spectral properties. Only the most red-shifted Chls, with emission beyond 730 nm, are efficiently quenched by P700 + , with rate constants of up to 6 ns -1 . The modeling results support the notion that structural and energetic disorder in Photosystem I can have a comparable or larger effect on the excitation dynamics than the geometric arrangement of Chls.

Phycobilisome (PBS) is a pigment-protein complex utilized by red algae and cyanobacteria in photosynthesis for light harvesting. A cyanobacterium Synechocystis sp. PCC 6803 contains PBS with a tricylindrical core built of allophycocyanin (APC) disks where six phycocyanin (PC) rods are attached. The top core cylinder is seemingly involved in attaching four PC rods and binding orange carotenoid protein (OCP) to quench excess of excitation energy. In this study, we have deleted the third linker domain (LD3) of ApcE subunit of PBS which assembles four APC discs into the top core cylinder. The mutation resulted in PBS with bicylindrical core, structurally comparable to the naturally existing PBS from Synechococcus 7942. Lack of LD3 and the top APC cylinder reduces the excitation energy transfer between PC and APC in the mutant. Moreover, these PBSs are more prone to light induced-photodamage and do not bind to the photoactivated orange carotenoid protein (OCP), a known PBS excitation quencher. These findings highlight the complex and elegant interplay between the PBS architecture and the functional efficiency, suggesting that in PBSs with naturally tri-cylindrical cores, the top cylinder has essential roles in recruiting the rods and proper binding of OCP., (© 2024 Wiley-VCH GmbH.)

A general one-step lateral flow immunochromatographic assay (LFIA) for the quantitative detection of triadimefon (TDF) and triadimenol (TDN) in fruit and vegetable samples was developed using time-resolved fluorescence microspheres (TRFM) as labels. A specific anti-triadimefon monoclonal antibody (mAb) was conjugated with TRFM to fabricate LFIA test strips. A time-resolved fluorometer as an LFIA reader was applied to obtain quantitative results and assess risk ranges for the LFIA test strips. Under the optimized experimental conditions, the limits of detection (LODs) in buffer/cucumbers/tomatoes/oranges were 0.046 ng/mL, 0.135 µg/kg, 1.047 µg/kg, and 5.811 µg/kg, respectively, which are ca. 1000 times lower than that of colloidal gold-labeled strips. The recovery in cucumber/tomato/orange samples was 109.4-116.7%, 87.7-110.9%, and 88.0-111.9%, respectively, indicating that the test strips had good reliability. Coupled with the easily customizable pretreatment procedures for various samples, the LFIA results were obtained within 18 min without the need for professional personnel or complicated equipment. TRFM-LFIA for TDF and TDN also shows remarkable specificity and precision. The test strips were also low-cost, portable, and convenient to use. These results indicate the test strips could be utilized as a novel strategy for on-site detection of TDF and TDN, which has the potential to expand and detect other pesticide or insecticide residues in food., (© 2024. The Author(s), under exclusive licence to Springer-Verlag GmbH Austria, part of Springer Nature.)

Amantadine (AMA), commonly used to treat viral infections in livestock and poultry, has been banned owing to its potential hazards to human well-being. To detect unauthorized AMA usage in livestock, we developed a polyclonal antibody with a high affinity for the specific recognition of AMA through a rational design based on a structure similar to AMA and revealed the availability of the hapten design by computational chemistry analysis. Using this antibody, we established a highly responsive time-resolved fluorescence immunochromatographic assay (TRFICA). The visual detection limit of the assay is 0.6 μg/kg, and the quantitative detection limit is 0.05 μg/kg. The TRFICA also showed good recovery rates ranging from 94.5 to 109.9%, with variability coefficients not exceeding 10%. The outcomes of undisclosed sample examinations aligned with those of HPLC-MS/MS analyses, indicating that this approach can function as an ideal screening and monitoring tool for detecting illegal AMA in chicken muscle.

Photosystem I (PSI) is a large membrane photosynthetic complex that harvests sunlight and drives photosynthetic electron transport. In both green algae and higher plants, PSI's ultrafast energy transfer and charge separation kinetics have been characterized. In contrast, it is not yet clear in Physcomitrella patens, even though moss is one of the earliest land plants and represents a critical stage in plant evolution. Here, we measured the time-resolved fluorescence of purified Pp PSI-LHCI at both room temperature (RT) and 77 K. Compared to the PSI kinetics of Arabidopsis thaliana at RT, we found that although the overall trapping time of Pp PSI-LHCI is nearly identical, ∼46 ps, their lifetimes at different wavelength regions differ. Specifically, Pp PSI-LHCI is slower in energy trapping below 720 nm but faster beyond. The slow-down of energy transfer between bulk chlorophylls (Chls, <720 nm) in Pp PSI-LHCI is probably because of the larger spatial gap between the PSI core and LHCI belt, and the acceleration of trapping at longer wavelength is most likely due to the lack of low-energy red-shifted Chls (red Chls). Indeed, time-resolved fluorescence results at 77 K revealed only three types of red Chls of 702 nm, 712 nm, and 720 nm in Pp PSI-LHCI but failed to detect the red Chls of 735 nm that present in LHCI in higher plants. Finally, we briefly discussed the evolutionary adaptations of PSI-LHCI in the context of red Chls from green algae to mosses and to land plants., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024. Published by Elsevier B.V.)

Digital histogram generation for time-resolved measurements with single-photon avalanche diode (SPAD) sensors requires the storage of many timestamp signals. This work presents a mixed-signal time-to-digital converter (TDC) that uses analog storage to achieve an area-efficient design that can be integrated in large SPAD arrays. Fabricated using a 150 nm CMOS process, the prototype occupies an area of only 18.3 µm × 36.5 µm, a notable size reduction compared to conventional designs. The experimental results demonstrated high performance, with an integral nonlinearity (INL) of 0.35/0.14 least significant bit (LSB) and a differential nonlinearity (DNL) of 0.14/-0.12 LSB. In addition, the proposed TDC can support the construction of histograms comprising up to 512 bins, making it an effective solution to accommodate a wide range of resolution requirements. Validated in a point-of-care (PoC) device for fluorescence lifetime measurements, it distinguished between lifetimes of approximately 4.1 ns, 3.6 ns and 80 ns with the Alexa Fluor (AF) 546 and 568 dyes and Quantum Dot (QD) 705, respectively. The analog storage design and area-efficient architecture offer a novel approach to integrating TDCs in SPAD-based systems, with potential applications in medical diagnostics and beyond.

Aggregation of microtubule-associated protein tau (MAPT/tau) into conformationally distinct fibrils underpins neurodegenerative tauopathies. Fluorescent probes (fluoroprobes), such as thioflavin T (ThT), have been essential tools for studying tau aggregation; however, most of them do not discriminate between amyloid fibril conformations (polymorphs). This gap is due, in part, to a lack of high-throughput methods for screening large, diverse chemical collections. Here, we leverage advances in protein adaptive differential scanning fluorimetry (paDSF) to screen the Aurora collection of 300+ fluorescent dyes against multiple synthetic tau fibril polymorphs. This screen, coupled with orthogonal secondary assays, revealed pan-fibril binding chemotypes, as well as fluoroprobes selective for subsets of fibrils. One fluoroprobe recognized tau pathology in ex vivo brain slices from Alzheimer's disease patients. We propose that these scaffolds represent entry points for development of selective fibril ligands and, more broadly, that high throughput, fluorescence-based dye screening is a platform for their discovery., Competing Interests: Competing Interests The authors have no competing interests to disclose.

A fully quantum, numerically accurate methodology is presented for the simulation of the exciton dynamics and time-resolved fluorescence of cavity-tuned two-dimensional (2D) materials at finite temperatures. This approach was specifically applied to a monolayer WSe2 system. Our methodology enabled us to identify the dynamical and spectroscopic signatures of polaronic and polaritonic effects and to elucidate their characteristic timescales across a range of exciton-cavity couplings. The approach employed can be extended to simulation of various cavity-tuned 2D materials, specifically for exploring finite temperature nonlinear spectroscopic signals.

Phycobilisome (PBS) is a large pigment-protein complex in cyanobacteria and red algae responsible for capturing sunlight and transferring its energy to photosystems (PS). Spectroscopic and structural properties of various PBSs have been widely studied, however, the nature of so-called complex-complex interactions between PBS and PSs remains much less explored. In this work, we have investigated the function of a newly identified PBS linker protein, ApcG, some domain of which, together with a loop region (PB-loop in ApcE), is possibly located near the PBS-PS interface. Using Synechocystis sp. PCC 6803, we generated an ApcG deletion mutant and probed its deletion effect on the energetic coupling between PBS and photosystems. Steady-state and time-resolved spectroscopic characterization of the purified ΔApcG-PBS demonstrated that ApcG removal weakly affects the photophysical properties of PBS for which the spectroscopic properties of terminal energy emitters are comparable to those of PBS from wild-type strain. However, analysis of fluorescence decay imaging datasets reveals that ApcG deletion induces disruptions within the allophycocyanin (APC) core, resulting in the emergence (splitting) of two spectrally diverse subgroups with some short-lived APC. Profound spectroscopic changes of the whole ΔApcG mutant cell, however, emerge during state transition, a dynamic process of light scheme adaptation. The mutant cells in State I show a substantial increase in PBS-related fluorescence. On the other hand, global analysis of time-resolved fluorescence demonstrates that in general ApcG deletion does not alter or inhibit state transitions interpreted in terms of the changes of the PSII and PSI fluorescence emission intensity. The results revealed yet-to-be discovered mechanism of ApcG-docking induced excitation energy transfer regulation within PBS or to Photosystems., Competing Interests: Declaration of competing interest The authors have no competing interests to declare that are relevant to the content of this article., (Copyright © 2024 Elsevier B.V. All rights reserved.)

Wines are complex mixtures of chemical compounds with broad and overlapping absorption and emission spectral features in the UV and visible spectral regions, making them challenging to study with conventional optical spectroscopic techniques. Multidimensional fluorescence spectroscopies correlate fluorescence spectra with other degrees of freedom, and have proven useful for studying complex molecular systems, offering a pathway for the analysis of wines utilising their inherent fluorescence. Here we employ steady-state excitation-emission matrix (EEM) and time-resolved fluorescence spectral measurements to investigate representative commercial white and red wine samples and a fluorescent 'model' wine base. Combining these multidimensional measurement methods provides information on the emission characteristics of the components that wines contain. This investigation illustrates the potential for multidimensional fluorescence techniques as diagnostic tools for the wine industry., (Creative Commons Attribution license.)

The alignment of chromophores plays a crucial role in determining the optoelectronic properties of materials. Such alignment can make interpretation of fluorescence anisotropy microscopy (FAM) images somewhat ambiguous. The time-resolved emission behaviour can also influence the fluorescence anisotropy. This is particularly the case when probing excitation energy migration between chromophores in a condensed phase. Ideally information concerning the chromophoric alignment, emission decay kinetics and fluorescence anisotropy can be recorded and correlated. We report on the use of polarised transmission imaging (PTI) coupled with both steady-state and time-resolved FAM to enable accurate identification of chromophoric alignment and morphology in thin films of a conjugated polydiarylfluorene. We show that the combination of these three imaging modes presents a comprehensive methodology for investigating the alignment and morphology of chromophores in thin films, particularly for accurately mapping the distribution of amorphous and crystalline phases within the thin films, offering valuable insights for the design and optimization of materials with enhanced optoelectronic performance., (Creative Commons Attribution license.)

The goal of this study is to create a highly sensitive time-resolved fluorescence lateral flow immunoassay (TRF-LFIA) capable of concurrently measuring glial fibrillary acidic protein (GFAP) and the N-terminal fragment of B-type natriuretic peptide precursor (NT-proBNP). This assay is designed as a diagnostic tool and aims to provide an algorithm for stroke management, specifically for distinguishing between Ischemic stroke (IS) and Hemorrhagic stroke (HS). However, LFIA to quantify simultaneous serum NT-proBNP and GFAP are not yet available. We have developed and validated a novel TRF-LFIA for the simultaneous quantitative detection of NT-proBNP and GFAP. The sensitivity and reproducibility of the immunoassay were significantly improved by employing specific monoclonal antibodies linked to europium nanoparticles (EuNPs) that specifically target NT-proBNP and GFAP. The detection area on the nitrocellulose membrane featured sandwich-style complexes containing two test lines for NT-proBNP and GFAP, and one Control line. The fluorescence intensity of these test lines and control line was measured using an in-house developed Exdia TRF-Plus analyzer. As proof-of-concept, we enrolled patients suspected of having a stroke who were admitted within a specific time frame (6 h). A small amount of clinical specimen (serum) was used. To optimize the LFIA, an EuNPs conjugated antibodies were investigated to improve the detection sensitivity and decrease the background signal as well shorten the detection time. The Exdia TRF-LFIA cartridge offers a wide linear dynamic detection range, rapid detection, high sensitivity, and specificity. The limit of detection was determined to be 98 pg/mL for NT-proBNP and 68 pg/mL for GFAP, with minimal cross-reactivity. There were 200 clinical human serum samples that were used to evaluate this platform with high correlation. By combining the results of NT-proBNP and GFAP, we formulated an algorithm for the clinical assessment of Ischemic Stroke (IS) and Hemorrhagic Stroke (HS). According to our proposed algorithm, the combination of GFAP and NT-proBNP emerged as the most effective biomarker combination for distinguishing between IS and HS. Exdia TRF-LFIA shows great potential as a supplemental method for in vitro diagnostics in the laboratory or in other point-of-care testing (POCT) applications. Its development substantially decreases the diagnosis time for IS and HS. The proposed algorithm not only minimizes treatment delays but also lowers medical costs for patients., (Copyright © 2024. Published by Elsevier B.V.)

Objectives: A major challenge in non-small cell lung cancer surgery is the occurrence of positive tumor margins. This may lead to the need for additional surgeries and has been linked to poor patient prognosis. This study aims to develop an in vivo surgical tool that can differentiate cancerous from noncancerous lung tissue at the margin., Methods: A time-resolved fluorescence and diffuse reflectance bimodal device was used to measure the lifetime, spectra, and intensities of endogenous fluorophores as well as optical properties of lung tissue. The tumor and fibrotic tissue data, each containing 36 samples, was obtained from patients who underwent surgical removal of lung tissue after being diagnosed with squamous carcinoma but before any other treatment was administered. The normal lung tissue data were obtained from nine normal tissue samples., Results: The results show a statistically significant difference between cancerous and noncancerous tissue. The results also show a difference in metabolic related optical properties between fibrotic and normal lung tissue samples., Conclusions: This work demonstrates the feasibility of a device that can differentiate cancerous and noncancerous lung tissue for patients diagnosed with squamous cell carcinoma., (© 2024 The Authors. Lasers in Surgery and Medicine published by Wiley Periodicals LLC.)

Jacalin, the jackfruit seed lectin, exhibits high specificity for the tumor-specific T-antigen and is used in various biomedical and biotechnological applications. Here, we report biophysical studies on the thermal unfolding of jacalin and the effect of pH and temperature on its secondary structure. Differential scanning calorimetric (DSC) studies revealed that native jacalin unfolds at ∼60 °C and that carbohydrate binding stabilizes the protein structure. Circular dichroism spectroscopic studies indicated that the secondary structure of jacalin remains mostly unaffected over pH 2.0-9.0, whereas considerable changes were observed in the tertiary structure. DSC experiments demonstrated that jacalin exhibits two overlapping transitions between pH 2 and 5, which could be attributed to dissociation of the tetrameric protein into subunits and their unfolding. Interestingly, only one transition between pH 6 and 9 was observed, suggesting that the subunit dissociation and unfolding occur simultaneously. While quenching of the protein intrinsic fluorescence by acrylamide increased significantly upon carbohydrate binding, quenching by succinimide is essentially unaffected. We attribute this difference to increased exposure of Trp-123 in the α-chain as it is involved in carbohydrate binding. Both acrylamide and succinimide gave biphasic Stern-Volmer plots, consistent with differential accessibility of the two tryptophan residues of jacalin to them., Competing Interests: Declaration of competing interest The authors declare the following financial interests/personal relationships which may be considered as potential competing interests: Musti J. Swamy reports financial support was provided by India Ministry of Science & Technology Department of Science and Technology. Musti J. Swamy reports financial support was provided by University of Hyderabad., (Copyright © 2024 Elsevier B.V. All rights reserved.)

Background: Fluorescence immunoassays are commonly employed for the detection of pathogenic bacteria as a means of ensuring food safety and preserving public health. However, the challenges such as poor photostability and background interference have limited their sensitivity and accuracy. The emergence of metal-organic frameworks (MOFs) as a label probe offers a promising solution for advancing fluorescence immunoassays owing to their tunable nature. Nonetheless, the low fluorescence efficiency of MOFs and the potential risk of dye leakage pose obstacles to achieving high detection sensitivity. Therefore, there exists a pressing need to fully utilize the potential of MOF composites in fluorescence immunoassays., Results: We explored the potential of glucose oxidase-encapsulated zeolitic imidazole framework-90 (GOx@ZIF-90) as a label probe to construct a time-resolved fluorescence immunoassay with amplified detection signal. This immunoassay involved functionalizing Fe 3 O 4 nanoparticle with porcine antibody to specifically capture and separate the target bacteria, Staphylococcus aureus (S. aureus). The captured S. aureus was then bound by GOx@ZIF-90 modified with vancomycin, resulting in a fluorescence response in the europium tetracycline (EuTc). The encapsulation of GOx in ZIF-90 provided a confinement effect that significantly enhanced the catalytic activity and stability of GOx. This led to a highly efficient conversion of glucose to H 2 O 2 , amplifying the fluorescence signal of EuTc. The immunoassay demonstrated a high sensitivity in detecting S. aureus, with a detection limit of 2 CFU/mL. We also obtained satisfactory results in milk samples. Attractively, the time-resolved detection mode of EuTc allowed the immunoassay to eliminate background fluorescence and enhance accuracy., Significance: This study not only presented a new method for detecting foodborne pathogens but also highlighted the potential of enzyme-encapsulated MOF composites as label probes in immunoassays, providing valuable insights for the design and fabrication of MOF composites for various applications., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2023 Elsevier B.V. All rights reserved.)

Lead (Pb 2+ ) is one of the most toxic heavy metals, and its environmental pollution and serious damages is a global concern. Therefore, it is necessary to develop effective sensing methods. This study describes a new strategy for the design of label-free competitive time-resolved fluorescent (TRF) aptasensor for detecting Pb 2+ ions. The sensing principle of this aptasensor is the competition between Pb 2+ and Tb 3+ ions to bind to the guanine/thymine-rich sequence (lead aptamer) and the sensitized luminescence of Tb 3+ by this sequence. The developed TRF aptasensor demonstrated a good linear detection range from 2.5 nM to 150 nM and a limit of detection (LOD) of 645 pM. In addition, the proposed TRF aptasensor has a high selectivity towards Pb 2+ , and it has also been successfully utilized to detect this ion in milk and human serum samples. This TRF aptasensor offers advantages such as short analysis time, simple operation, low cost, being label-free, and surpassing the interference of background fluorescence of biological samples due to its TRF characteristic. In this paper, we propose a Tb 3+ /guanine/thymine-rich sequence system for designing different aptasensors against diverse targets by applying a proper guanine-rich sequence., Competing Interests: Declaration of Competing Interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2023 Elsevier B.V. All rights reserved.)

Fluorescence techniques have been widely used to shed light over the structure-function relationship of potassium channels for the last 40-50 years. In this chapter, we describe how a Förster resonance energy transfer between identical fluorophores (homo-FRET) approach can be applied to study the gating behavior of the prokaryotic channel KcsA. Two different gates have been described to control the K + flux across the channel's pore, the helix-bundle crossing and the selectivity filter, located at the opposite sides of the channel transmembrane section. Both gates can be studied individually or by using a double-reporter system. Due to its homotetrameric structural arrangement, KcsA presents a high degree of symmetry that fulfills the first requisite to calculate intersubunit distances through this technique. The results obtained through this work have helped to uncover the conformational plasticity of the selectivity filter under different experimental conditions and the importance of its allosteric coupling to the opening of the activation (inner) gate. This biophysical approach usually requires low protein concentration and presents high sensitivity and reproducibility, complementing the high-resolution structural information provided by X-ray crystallography, cryo-EM, and NMR studies., (© 2024. The Author(s), under exclusive license to Springer Science+Business Media, LLC, part of Springer Nature.)

Circular permutation (CP) is a technique by which the primary sequence of a protein is rearranged to create new termini. The connectivity of the protein is altered but the overall protein structure generally remains unperturbed. Understanding the effect of CP can help design robust proteins for numerous applications such as in genetic engineering, optoelectronics, and improving catalytic activity. Studies on different protein topologies showed that CP usually affects protein stability as well as unfolding rates. Though a significant number of proteins contain metals or other cofactors, reports of metalloprotein CPs are rare. Thus, we chose a bacterial metalloprotein, azurin, and its CP within the metal-binding site (cpF114). We studied the stabilities, folding, and unfolding rates of apo- and Zn 2+ -bound CP azurin using fluorescence and circular dichroism. The introduced CP had destabilizing effects on the protein. Also, the folding of the Zn 2+ -CP protein was much slower than that of the Zn 2+ -WT or apo-protein. We compared this study to our previously reported azurin-cpN42, where we had observed an equilibrium and kinetic intermediate. cpF114 exhibits an apparent two-state equilibrium unfolding but has an off-pathway kinetic intermediate. Our study hinted at CP as a method to modify the energy landscape of proteins to alter their folding pathways. WT azurin, being a faster folder, may have evolved to optimize the folding rate of metal-bound protein compared to its CPs, albeit all of them have the same structure and function. Our study underscores that protein sequence and protein termini positions are crucial for metalloproteins. TOC Figure. (Top) Zn 2+ -azurin WT structure (PDB code: 1E67) and 2-D topology diagram of Zn 2+ -cpF114 azurin. (Bottom) Cartoon diagram representing folding (red arrows) and unfolding (blue arrows) of apo- and Zn 2+ - WT and cpF114 azurins. The width of the arrows represents the rate of the corresponding processes., (© 2023. The Author(s), under exclusive licence to Society for Biological Inorganic Chemistry (SBIC).)

Phycobilisomes serve as a light-harvesting antenna of both photosystem I (PSI) and II (PSII) in cyanobacteria, yet direct energy transfer from phycobilisomes to PSI is not well documented. Here we recorded picosecond time-resolved fluorescence at wavelengths of 605-760 nm in isolated photosystem I (PSI), phycobilisomes and intact cells of a PSII-deficient mutant of Synechocystis sp. PCC 6803 at 77 K to study excitation energy transfer and trapping. By means of a simultaneous target analysis of the kinetics of isolated complexes and whole cells, the pathways and dynamics of energy transfer in vitro and in vivo were established. We establish that the timescale of the slowest equilibration between different terminal emitters in the phycobilisome is ≈800 ps. It was estimated that the terminal emitter in about 40% of the phycobilisomes transfers its energy with a rate constant of 42 ns -1 to PSI. This energy transfer rate is higher than the rates of equilibration within the phycobilisome - between the rods and the core or between the core cylinders - and is evidence for the existence of specific phycobilisome-PSI interactions. The rest of the phycobilisomes remain unconnected or slowly transferring energy to PSI., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest. The author(s) declared that they were an editorial board member of Frontiers, at the time of submission. This had no impact on the peer review process and the final decision., (Copyright © 2023 van Stokkum, Akhtar, Biswas and Lambrev.)

The fluorescence assay is one of the popular methods that is applied for detection of different targets. However, this method may show low sensitivity and high background in biological samples due to the natural fluorescence of different compounds in complicated samples. In addition, it inevitably affects the detection results accuracy. A fundamental solution to this problem is the use of the time-resolved fluorescence technique (TRF). The main component of this technique is the use of long fluorescence lifetime reagents. In this review, various time-resolved fluorescent reagents such as complexes of lanthanide ions, lanthanide-doped inorganic nanoparticles; Mn-doped ZnS quantum dots (QDs) and pyrene excimer are introduced. Moreover, TRF sensors, especially TRF aptasensors (DNA-based sensors) are discussed. This review will give new ideas for researchers to develop novel high-sensitive TRF sensors that can remove or decrease background fluorescence and use them for the detection of various targets in complicated samples without treatment., (© 2023. The Author(s), under exclusive licence to Springer Science+Business Media, LLC, part of Springer Nature.)

Interaction of an antibody with its FcγR plays an important role in effector functions such as antibody-dependent cell-mediated cytotoxicity (ADCC). Nowadays altered ADCC activity of an antibody can be achieved by utilizing an effective glyco-engineering strategy, which often involves changes of sugar moieties in Fc part of the antibody, thereby affecting its receptor binding with effector cells. We aimed to construct a cell-based time-resolved fluorescence (TRF) assay for the evaluation of ADCC activity triggered by the antibody drug Trastuzumab (anti-HER2) and T-DM1. The assay was initiated by incubating 2,2':6',2 "-Terpyridine-6,6"-dicarboxylic acid (TDA)-labeled target SK-BR3 cells with the testing antibodies and engineered NK-92 effector cells. After incubation, the target cells were lysed to detect TDA released into the supernatant. Together with added Eu, the TDA in the supernatant formed a stable chelate of EuTDA with high-intensity fluorescence. The ADCC activity was then determined by measuring the fluorescence of EuTDA. Consequently, the method demonstrated good accuracy, precision, linearity, and specificity over methodological assessment and compared well with the Luciferase release assay in terms of the agreement of the achieved results. Using the developed assay, we evaluated the ADCC activity of two glyco-engineered anti-HER-2 antibody-drug conjugates (ADCs) and the results showed that antibody Fc glycosylation modifications influenced antibody ADCC activity to varying degrees. In conclusion, the present assay is able to accurately assess the ADCC activity induced by Trastuzumab (anti-HER2) and T-DM1, and a similar methodology can be applied to other therapeutic antibodies during drug development to help screen for antibodies with desirable ADCC activity., Competing Interests: Declaration of Competing Interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2023 Elsevier B.V. All rights reserved.)

Kidney disorders are a rising global health issue, necessitating early diagnosis for effective treatment. Creatinine, a metabolic waste product from muscles, serves as an ideal biomarker for kidney damage. The existing optical methods for creatinine detection often involve labor-intensive synthesis processes and present challenges with the aqueous solubility and sensitivity to experimental variations. In this study, we introduce a straightforward fluorescence "turn-on" ratiometric sensor system for creatinine detection in aqueous media with a limit of detection of 0.5 μM. The sensor is based on sulfated-β-cyclodextrin (SCD)-templated H-aggregate of a commercially available, ultrafast rotor dye thioflavin-T (ThT). The Al 3+ ion-induced dissociation of ThT-SCD aggregates, followed by reassociation upon creatinine addition, generates a detectable signal. The modulation of monomer/aggregate equilibrium due to the disassembly/reassembly of the ThT-SCD system under Al 3+ /creatinine influence serves as the optimal strategy for ratiometric creatinine detection in aqueous media. Our sensor framework offers several advantages: utilization of the readily available dye ThT, which eliminates the need for a laborious synthesis of custom fluorescent probes; ratiometric sensing, which improves quantitative analysis accuracy; and compatibility with complex aqueous media. The sensor's practical utility has been successfully demonstrated in artificial urine samples. In summary, our sensor system represents a significant advancement in the rapid, selective, and sensitive detection of the clinically crucial bioanalyte creatinine, offering potential benefits for the early diagnosis and management of kidney disorders.

Water treatment in photocatalytic membrane reactors (PMR) holds great promise for removing micropollutants from aquatic environments. Organic matter (OM) that is present in any water matrix may significantly interfere with the degradation of steroid hormone (SH) micropollutants in PMRs. In this study, the interference of various OM types, humic acid (HA), Australian natural organic matter (AUS), worm farm extract (WF), tannic acid (TA), and gallic acid (GA) with the SH degradation at its environmentally relevant concentration (100 ng/L) in a flow-through PMR equipped with a polyethersulphone-titanium dioxide (PES-TiO 2 ) membrane operated under UV light (365 nm) was investigated. Results of this study showed that OM effects are complex and depend on OM type and concentration. The removal of β-estradiol (E2) was enhanced by HA at its levels below 5 mgC/L while the enhancement was abated at higher HA concentrations. The E2 removal was inhibited by TA, and GA, while no significant interference observed for AUS, and WF. The data demonstrated diverse roles of OM that acts in PMRs as a light screening agent, a photoreactive species scavenger, an adsorption alteration trigger, and a photosensitizer. The time-resolved fluorescence measurement showed that HA, acting as a photosensitizer, promoted the sensitization of TiO 2 by absorbing light energy and transferring energy/electron to the TiO 2 substrate. This pathway dominated the mechanism of the enhanced E2 degradation by HA. The favorable effect of HA was augmented as increasing the light intensity from 0.5 to 10 mW/cm 2 and was weakened at higher light intensities due to the increased scavenging reactions and the limited amount of HA. This work clarifies the underlying mechanism of the OM interference on photocatalytic degradation of E2 by the PES-TiO 2 PMR., Competing Interests: Declaration of Competing Interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2023. Published by Elsevier Ltd.)

The fusion of tetrapyrroles with aromatic heterocycles constitutes a useful tool for manipulating their opto-electronic properties. In this work, the synthesis of naphthodithiophene-fused porphyrins was achieved through a Heck reaction-based cascade of steps followed by the Scholl reaction. The naphthodithiophene-fused porphyrins display a unique set of optical and electronic properties. Fusion of the naphtho[2,1-b:3,4-b']dithiophene to porphyrin (F2VTP) leads to a ~20% increase in the fluorescence lifetime, which is accompanied, unexpectedly, by a more than two-fold drop in the emission quantum yield (ϕ=0.018). In contrast, fusion of the isomeric naphtho[1,2-b:4,3-b']dithiophene to porphyrin (F3VPT) results in a ~1.5-fold increase in the fluorescence quantum yield (ϕ=0.13) with a concomitant ~30 % increase in the fluorescence lifetime. This behavior suggests that fusion of the porphyrin with the naphthodithiopheno-system mainly affects the radiative rate constant in the Q-state deactivation pathway, where the effects of the isomeric naphtho[2,1-b:3,4-b']dithiophene- versus naphtho[1,2-b:4,3-b']dithiophene-fusion are essentially the opposite. Interestingly, nucleus-independent chemical shifts analysis revealed a considerable difference between the aromaticities of these two isomeric systems. Our results demonstrate that subtle structural differences in the fused components of the porphyrin can be reflected in rather significant differences between the photophysical properties of the resulting systems., (© 2023 Wiley-VCH GmbH.)

The development of an accurate and sensitive sensor for detecting amyloid plaques, which are responsible for many protein disorders like Alzheimer's disease, is crucial for early diagnosis. Recently, there has been a notable increase in the development of fluorescence probes that exhibit emission in the red region (>600 nm), aiming to effectively tackle the challenges encountered when working with complex biological matrices. In the current investigation, a hemicyanine-based probe, called LDS730, has been used for the sensing of amyloid fibrils, which belong to the Near-Infrared Fluorescence (NIRF) family of dyes. NIRF probes provide higher precision in detection, prevent photo-damage, and minimize the autofluorescence of biological specimens. The LDS730 sensor emits in the near-infrared region and shows a 110-fold increase in fluorescence turn-on emission when bound to insulin fibrils, making it a highly sensitive sensor. The sensor has an emission maximum of ~710 nm in a fibril-bound state, which shows a significant red shift along with a Stokes' shift of ~50 nm. The LDS730 sensor also displays excellent performance in the complicated human serum matrix, with a limit of detection (LOD) of 103 nM. Molecular docking calculations suggest that the most likely binding location of LDS730 in the fibrillar structure is the inner channels of amyloid fibrils along its long axis, and the sensor engages in several types of hydrophobic interactions with neighboring amino acid residues of the fibrillar structure. Overall, this new amyloid sensor has great potential for the early detection of amyloid plaques and for improving diagnostic accuracy., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2023 Elsevier B.V. All rights reserved.)

Titanium-based catalysis in single electron transfer (SET) steps has evolved into a versatile approach for the synthesis of fine chemicals and first attempts have recently been made to enhance its sustainability by merging it with photo-redox (PR) catalysis. Here, we explore the photochemical principles of all-Ti-based SET-PR-catalysis, i.e. in the absence of a precious metal PR-co-catalyst. By combining time-resolved emission with ultraviolet-pump/mid-infrared-probe (UV/MIR) spectroscopy on femtosecond-to-microsecond time scales we quantify the dynamics of the critical events of entry into the catalytic cycle; namely, the singlet-triplet interconversion of the do-it-all titanocene(IV) PR-catalyst and its one-electron reduction by a sacrificial amine electron donor. The results highlight the importance of the PR-catalyst's singlet-triplet gap as a design guide for future improvements., (© 2023 The Authors. Angewandte Chemie International Edition published by Wiley-VCH GmbH.)

Hydroxychloroquine (HCQ), a quinoline based medicine is commonly used to treat malaria and autoimmune diseases such as rheumatoid arthritis. Since, human serum albumin (HSA) serves as excipient for vaccines or therapeutic protein drugs, it is important to understand the effect of HCQ on the structural stability of HSA. In this study, the binding mechanism of HCQ and their effect on stability of HSA have been studied using various spectroscopic techniques and molecular dynamic simulation. The UV-VIS results confirmed the strong binding of HCQ with HSA. The calculated thermodynamics parameters confirmed that binding is spontaneous in nature and van der Waals forces and hydrogen bonding are involved in the binding system which is also confirmed by molecular docking results. The steady-state fluorescence confirms the static quenching mechanism in the interaction system, which was further validated by time-resolved fluorescence. The synchronous fluorescence confirmed the more abrupt binding of HCQ with tryptophan residue of HSA compared to Tyr residue of HSA. Isothermal titration calorimetry (ITC) was done to validate the thermodynamics parameters of HSA-HCQ complex in one experiment, supporting the values obtained from the spectroscopic techniques. The circular dichroism (CD) demonstrated that the HCQ affected the secondary structure of HSA protein by reducing their α-helical content. The docking and molecular dynamic simulation results further helped in understanding the effect of HCQ on conformational changes of HSA. Overall, present work defined the physicochemical properties and interaction mechanism of HCQ with HSA that have extensively been elucidated by both in vitro and in silico approaches.Communicated by Ramaswamy H. Sarma.

Photosystem II in oxygenic organisms is a large membrane bound rapidly turning over pigment protein complex. During its biogenesis, multiple assembly intermediates are formed, including the CP43-preassembly complex (pCP43). To understand the energy transfer dynamics in pCP43, we first engineered a His-tagged version of the CP43 in a CP47-less strain of the cyanobacterium Synechocystis 6803. Isolated pCP43 from this engineered strain was subjected to advanced spectroscopic analysis to evaluate its excitation energy dissipation characteristics. These included measurements of steady-state absorption and fluorescence emission spectra for which correlation was tested with Stepanov relation. Comparison of fluorescence excitation and absorptance spectra determined that efficiency of energy transfer from β-carotene to chlorophyll a is 39 %. Time-resolved fluorescence images of pCP43-bound Chl a were recorded on streak camera, and fluorescence decay dynamics were evaluated with global fitting. These demonstrated that the decay kinetics strongly depends on temperature and buffer used to disperse the protein sample and fluorescence decay lifetime was estimated in 3.2-5.7 ns time range, depending on conditions. The pCP43 complex was also investigated with femtosecond and nanosecond time-resolved absorption spectroscopy upon excitation of Chl a and β-carotene to reveal pathways of singlet excitation relaxation/decay, Chl a triplet dynamics and Chl a → β-carotene triplet state sensitization process. The latter demonstrated that Chl a triplet in the pCP43 complex is not efficiently quenched by carotenoids. Finally, detailed kinetic analysis of the rise of the population of β-carotene triplets determined that the time constant of the carotenoid triplet sensitization is 40 ns., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2023 Elsevier B.V. All rights reserved.)

The detection of heavy metals in food is beneficial to public health. Herein, a new time-resolved fluorescent immunosensor based on the Förster resonance energy transfer (FRET) was proposed to rapidly detect a cadmium ion (Cd 2+ ). After coupling with an antigen and a monoclonal antibody (mAb), respectively, the synthesized europium (III)-chelate-doped polystyrene time-resolved fluorescence microsphere-antigen (Eu-antigen) and gold nanoflowers-mAb (GNFs-mAb) interacted by antigen-antibody combination to quench fluorescence through FRET. Under optimal conditions, the Eu-GNF immunosensing platform provided a quick response to Cd 2+ within 30 min. The limit of detection was 0.29 ng/mL with a linear range of 1-500 ng/mL. This immunosensor was further validated via graphite furnace atomic absorption spectrometry and exhibited satisfactory recovery for the detection of Cd 2+ in four kinds of tea samples (98.82-108.01%). Thus, this strategy provides a good paradigm for the rapid and high-throughput detection of heavy metals in field testing.

Benzo[a]pyrene is a widespread environmental pollutant and a strong carcinogen. It is important to understand its bio-toxicity and degradation mechanism. Herein, we studied the excited state dynamics of benzo[a]pyrene by using time-resolved fluorescence and transient absorption spectroscopic techniques. For the first time, it is identified that benzo[a]pyrene in its singlet excited state could react with oxygen, resulting in fluorescence quenching. Additionally, effective intersystem crossing can occur from its singlet state to the triplet state. Furthermore, the interaction between the excited benzo[a]pyrene and ct-DNA can be observed directly and charge transfer between benzo[a]pyrene and ct-DNA may be the reason. These results lay a foundation for further understanding of the carcinogenic mechanism of benzo[a]pyrene and provide insight into the photo-degradation mechanism of this molecule.

Mechanoluminescence (ML) has received widespread attention because of potential application in stress sensors and imaging. However, pursuing highly efficient ML remains a challenge due to multifactorial limitations such as pressure and loading rate. Here, we systematically investigate pressure- and rate-dependent ML in Mn 2+ and Eu 3+ co-doped ZnS in a gigapascal pressure range by using a high-pressure dynamic diamond anvil cell and microsecond time-resolved fluorescent methods and demonstrate the giant tunability in both ML efficiency and wavelength. Compressed from ambient pressure to 11 GPa at different compression rates, ZnS: Mn 2+ , Eu 3+ exhibits a volcano shape in ML emission efficiency with an optimum at ∼3.5 GPa and ∼211.1 GPa/s, at least 1000-fold higher than that measured in the MPa range. The pressure-dependent ML is accompanied with a tunable yellow-to-red emission color change. A combination of high-pressure X-ray diffraction and photoluminescence measurements reveals that the pressure- and rate-dependent ML behavior derives from pressure-induced strengthening of the crystal piezoelectric field and enhanced interaction between the host lattice and doped ions with a significant change of the energy level of the Mn ion. Significantly, the highly efficient ML of ZnS: Mn 2+ , Eu 3+ at the GPa level is reproducible under a compression-decompression process and can be manipulated on a micron scale, implying great potential in mechanical-optical energy conversion and application in dynamic pressure imaging, stress sensors, and multicolor displays.

Excited-state chemistry relies on the communication between molecules, making it a crucial aspect of the field. One important question that arises is whether intermolecular communication and its rate can be modified when a molecule is confined. To explore the interaction in such systems, we investigated the ground and excited states of 4'- N , N -diethylaminoflavonol (DEA3HF) in an octa acid-based (OA) confined medium and in ethanolic solution, both in the presence of Rhodamine 6G (R6G). Despite the observed spectral overlap between the flavonol emission and the R6G absorption, as well as the fluorescence quenching of the flavonol in the presence of R6G, the almost constant fluorescence lifetime at different amounts of R6G discards the presence of FRET in the studied systems. Steady-state and time-resolved fluorescence indicate the formation of an emissive complex between the proton transfer dye encapsulated within water-soluble supramolecular host octa acid (DEA3HF@(OA) 2 ) and R6G. A similar result was observed between DEA3HF:R6G in ethanolic solution. The respective Stern-Volmer plots corroborate with these observations, suggesting a static quenching mechanism for both systems.

Excited state intramolecular proton transfer (ESIPT) dynamics of the o -hydroxy analogs of the green fluorescent protein (GFP) chromophore have been investigated by time-resolved spectroscopies and theoretical calculations. These molecules comprise an excellent system to investigate the effect of electronic properties on the energetics and dynamics of ESIPT and to realize applications in photonics. Time-resolved fluorescence with high enough resolution was employed to record the dynamics and the nuclear wave packets in the excited product state exclusively in conjunction with quantum chemical methods. The ESIPT are ultrafast occurring in 30 fs for the compounds employed in this work. Although the ESIPT rates are not affected by the electronic properties of the substituents suggesting barrierless reaction, the energetics, their structures, subsequent dynamics following ESIPT, and possibly the product species are distinct. The results attest that fine tuning of the electronic properties of the compounds may modify the molecular dynamics of ESIPT and subsequent structural relaxation to achieve brighter emitters with broad tuning capabilities.

Herein, we have shown the interaction of levofloxacin (LVF) with two imidazolium based ionic liquids (ILs), 1-butly-3-methylimidazolium chloride ([Bmim][Cl]) and 1-decyl-3-methylimidazolium chloride ([Dmim][Cl]) by utilising spectroscopic techniques along with computational approach. Both [Bmim][Cl] and [Dmim][Cl] quenched the fluorescence emission of LVF suggesting complex formation between ILs and the drug. The steady-state and time-resolve fluorescence studies revealed that the quenching of fluorescence emission of LVF in the presence of [Bmim][Cl] and [Dmim][Cl], which signified the non-fluorescent complex formation between LVF and ILs. The complex formation between LVF and ILs were also validated by the UV-visible spectroscopy method. The cyclic voltammetry (CV) results further suggest the strong interaction between LVF and ILs. The estimated binding constant (K b ) and free energy change (ΔG) parameters shows the substantial binding of LVF with both the ILs and spontaneous in nature. The value suggested that LVF have stronger binding with [Dmim][Cl] than [Bmim][Cl]. Further, in order to support the results classical density functional theory (DFT) model was performed. The DFT calculations were utilized to explore the 3D structure and the molecular orbitals (HOMO and LUMO) of ILs, LVF and their complexes using Gaussian 09 software. The aggregate size (D h ) and zeta potential of ILs and IL-drug complexes were determined by dynamic light scattering (DLS) and zeta potential in aqueous medium., Competing Interests: Declaration of Competing Interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2022 Elsevier B.V. All rights reserved.)

Vibrio parahaemolyticus is a common foodborne pathogen in seafood, which often causes seafood borne bacterial gastroenteritis or food poisoning. Thermostable direct hemolysin (TDH) is considered to be one of the main virulence factors involved in this pathogen. The most clinical V. parahaemolyticus isolates produce TDH. Therefore, high sensitivity and specificity detection of TDH are of great significance for food safety and early diagnosis of diseases caused by V. parahaemolyticus. In this study, we developed a rapid, sensitive immunochromatographic test paper assay for the quantitative detection of TDH in seafood samples using time-resolved fluorescence techniques. First, we completed the preparation of fluorescent detection antibodies by coupling lanthanide fluorescent nanospheres with homemade high-affinity polyclonal antibodies based on the principle of the double-antibody sandwich. The lanthanide fluorescent nanospheres used in this study are characterized by a large stokes shift and a long fluorescence lifetime, which effectively reduces background noise and improves detection sensitivity. In addition, the method can be completed within 15 min for the detection of TDH, has a detection limit below 50 ng/mL and good linearity in the range of 50-5000 ng/mL. Moreover, it has good specificity and no cross-reactivity with Vibrio vulnificus hemolysin (VVH), Clostridium perfringens α toxin (CPA) or C. perfringens ε toxin (ETX). Finally, the sensitivity of this method was unchanged when the three simulated samples of Patinopecten yessoensis, Ruditapes philippinarum, and Scapharca broughtonii tested, indicating that the method is not affected by samples in a complex matrix. In conclusion, this study establishes a practical new method for on-site rapid detection of TDH, which is easy to operate, fast response, easy to carry and can be implemented under the field conditions without expensive equipment and professional person., (Copyright © 2022 The Author(s). Published by Elsevier Inc. All rights reserved.)

The co-facially stacked dyes on semiconductor films serve as an alternative model to elucidate the photo-driven exciton dynamics occurring in a molecular assembly. In this study, we report the unique emission properties of coumarin dye adsorbed on the surface of the semiconductor film, measured by ultrafast time-resolved fluorescence. When a rigid coumarin derivative, 7-hydroxycoumarin-3-carboxylic acid (OHCCA), is anchored on the Al 2 O 3 film, the dye manifests dual emissions from the two lowest excited states. Various anchoring modes of a carboxylic acid group on the Al 2 O 3 surface are invoked to account for the unusual emission process. Additionally, we identified characteristic transition dipole interactions in the well-stacked dye aggregates, which leads to discernible excitonic splitting in the electronic transitions. Femtosecond time-resolved fluorescence reveals that the excimer formation in the aggregate occurs with the time constant of 550 fs. Picosecond time-resolved emission spectra confirm the subsequent structural relaxations of the nascent excimer. The enhanced transition dipole via the electronic coupling between OHCCA and metal oxide can be responsible for the dual emission and the ultrafast excimer formation.

Histamine produced via the secretion of histidine decarboxylase by the bacteria in fish muscles is a toxic biogenic amine and of significant concern in food hygiene, since a high intake can cause poisoning in humans. This study proposed a fluorometric and colorimetric dual-mode specific method for the detection of histamine in fish, based on the fluorescence labeling of a histamine specific aptamer via the quenching and optical properties of gold nanoparticles (AuNPs). Due to the fluorescence resonance energy transfer phenomenon caused by the proximity of AuNPs and NaYF4:Ce/Tb, resulting in the quenching of the fluorescence signal in the detection system, the presence of histamine will compete with AuNPs to capture the aptamer and release it from the AuNP surface, inducing fluorescence recovery. Meanwhile, the combined detection of the two modes showed good linearity with histamine concentration, the linear detection range of the dual-mode synthesis was 0.2-1.0 μmol/L, with a detection limit of 4.57 nmol/L. Thus, this method has good selectivity and was successfully applied to the detection of histamine in fish foodstuffs with the recoveries of 83.39~102.027% and 82.19~105.94% for Trichiurus haumela and Thamnaconus septentrionalis , respectively. In addition, this method was shown to be simple, rapid, and easy to conduct. Through the mutual verification and combined use of the two modes, a highly sensitive, rapid, and accurate dual-mode detection method for the analysis of histamine content in food was established, thereby providing a reference for the monitoring of food freshness.

Many proteins form amyloid fibrils only under conditions when the probability of transition from a native (structured, densely packed) to an intermediate (labile, destabilized) state is increased. It implies the assumption that some structural intermediates are more convenient for amyloid formation than the others. Hence, if a mutation affects the protein folding pathway, one should expect that this mutation could affect the rate of amyloid formation as well. In the current work, we have compared the effects of amino acid substitutions of bovine carbonic anhydrase II on its unfolding pathway and on its ability to form amyloids at acidic pH and an elevated temperature. Wild-type protein and four mutant forms (L78A, L139A, I208A, and M239A) were studied. We analyzed the change of the protein unfolding pathway by the time-resolved fluorescence technique and the process of amyloid formation by thioflavin T fluorescence assay and electron microscopy. It was revealed that I208A substitution accelerates amyloid formation and affects the structure of the late (molten globule-like)-intermediate state of carbonic anhydrase, whereas the other mutations slow down the growth of amyloids and have either no effect on the unfolding pathway (L78A, L139A) or alter the conformational states arising at the early unfolding stage (M239A).

Despite the enormous environmental damage caused by plastic waste, it makes up over one-third of globally produced plastics. Polyethylene (PE) wastes have low recycling but high production rates. Towards the construction of ionic solar cells from PE, the present work describes the loading of a bioactive photoacid phycocyanobilin (PCB) dye from the pigment of Spirulina blue-green algae (as a natural resource) on low-density polyethylene (LDPE) plastic film. Dyeing was confirmed by X-ray photoelectron spectroscopy (XPS). Upon excitation of the Soret-band (400 nm), the photoluminescence (PL) spectra of PCB in neat solvents revealed two prominent emission peaks at 450-550 and 600-700 nm. The first band assigned to bilirubin-like (PCB BR ) species predominated the spectral profile in the highly rigid solvent glycerol and upon loading 0.45 % ( w / w ) of the dye on plastic. The photoluminescence excitation (PLE) spectra of PCB for the second region (Q-band) at 672 nm in the same solvents confirmed the ground state heterogenicity previously associated with the presence of PCB A (neutral), PCB B (cationic), and PCB C (anionic) conformers. Time-resolved photoluminescence (TRPL) measurements induced via excitation of all PCB species at 510 nm in methanol revealed three-lifetime components with τ 1 = ~0.1 ns and τ 2 = ~2 ns associated with PCB BR species and τ 3 = ~5 ns pertinent to the long-living photoproduct X*. Decay-associated spectra (DAS) analysis of the photoluminescence transient spectra of the final dyed films in the solid-state confirmed the improved generation of the long-living photoproduct as manifested in a significant increase in the PL intensity (~100-fold) and lifetime value (~90 ns) in the Q-region upon loading 6.92 % ( w / w ) of the dye on plastic. The photoproduct species were presumably assigned to the deprotonated PCB species, suggesting improved ionic mobility. The potential implementation of the PCB-sensitized PE solid wastes for the fabrication of ionic solar cells is discussed.

Plants and algae are faced with a conundrum: harvesting sufficient light to drive their metabolic needs while dissipating light in excess to prevent photodamage, a process known as nonphotochemical quenching. A slowly relaxing form of energy dissipation, termed qH, is critical for plants' survival under abiotic stress; however, qH location in the photosynthetic membrane is unresolved. Here, we tested whether we could isolate subcomplexes from plants in which qH was induced that would remain in an energy-dissipative state. Interestingly, we found that chlorophyll (Chl) fluorescence lifetimes were decreased by qH in isolated major trimeric antenna complexes, indicating that they serve as a site for qH-energy dissipation and providing a natively quenched complex with physiological relevance to natural conditions. Next, we monitored the changes in thylakoid pigment, protein, and lipid contents of antenna with active or inactive qH but did not detect any evident differences. Finally, we investigated whether specific subunits of the major antenna complexes were required for qH but found that qH was insensitive to trimer composition. Because we previously observed that qH can occur in the absence of specific xanthophylls, and no evident changes in pigments, proteins, or lipids were detected, we tentatively propose that the energy-dissipative state reported here may stem from Chl-Chl excitonic interaction., Competing Interests: Conflict of interest The authors declare that they have no conflicts of interest with the contents of this article., (Copyright © 2022 The Authors. Published by Elsevier Inc. All rights reserved.)

Light-harvesting antennas in photosynthesis capture light energy and transfer it to the reaction centers (RCs) where photochemistry takes place. The sustainable growth of the reef-building corals relies on a constant supply of the photosynthates produced by the endosymbiotic dinoflagellate, belonging to the family of Symbiodiniaceae. The antenna system in this group consists of the water-soluble peridinin-chlorophyll a-protein (PCP) and the intrinsic membrane chlorophyll a-chlorophyll c 2 -peridinin protein complex (acpPC). In this report, a nonameric acpPC is reported in a dinoflagellate, Fugasium kawagutii (formerly Symbiodinium kawagutii sp. CS-156). We found that extensive biochemical purification altered the oligomerization states of the initially isolated nonameric acpPC. The excitation energy transfer pathways in the acpPC nonamer and its variants were studied using time-resolved fluorescence and time-resolved absorption spectroscopic techniques at 77 K. Compared to the well-characterized trimeric acpPC, the nonameric acpPC contains an 11 nm red-shifted terminal energy emitter and substantially altered excited state lifetimes of Chl a. The observed energetic overlap of the fluorescence terminal energy emitters with the absorption of RCs is hypothesized to enable efficient downhill excitation energy transfer. Additionally, the shortened Chl a fluorescence decay lifetime in the oligomeric acpPC indicate a protective self-relaxation strategy. We propose that the highly-oligomerized acpPC nonamer represents an intact functional unit in the Symbiodiniaceae thylakoid membrane. They perform efficient excitation energy transfer (to RCs), and are under manageable regulations in favor of photoprotection., (© 2022. The Author(s), under exclusive licence to Springer Nature B.V.)

The ultrafast dynamics of triplet excitons and polarons in hexaphenyl film was investigated by time-resolved fluorescence and femtosecond transient absorption techniques under various excitation photon energies. Two distinct pathways of triplet formation were clearly observed. Long-lived triplet states are populated within 4.5 ps via singlet fission-intersystem crossing, while the short-lived triplet states (1.5 ns) are generated via singlet fission from vibrational electronic states. In the meantime, polarons were formed from hot excitons on a timescale of <30 fs and recombined in ultrafast lifetime (0.37 ps). In addition, the characterization of hexaphenyl film suggests the morphologies of crystal and aggregate to wide applications in organic electronic devices. The present study provides a universally applicable film fabrication in hexaphenyl system towards future singlet fission-based solar cells.

Background: The spread of COVID-19 worldwide caused by the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) has necessitated efficient, sensitive diagnostic methods to identify infected people. We report on the development of a rapid 15-minute time-resolved fluorescent (TRF) lateral flow immunochromatographic assay for the quantitative detection of the SARS-CoV-2 spike protein receptor-binding domain (S1-RBD)., Objectives: Our objective was to develop an efficient method of detecting SARS-CoV-2 within 15 min of sample collection., Methods: We constructed and evaluated a portable, disposable lateral flow device, which detected the S1-RBD protein directly in nasopharyngeal swab samples. The device emits a fluorescent signal in the presence of S1-RBD, which can be captured by an automated TRF instrument., Results: The TRF lateral flow assay signal was linear from 0 to 20 ng/ml and demonstrated high accuracy and reproducibility. When evaluated with clinical nasopharyngeal swabs, the assay was performed at >80% sensitivity, >84% specificity, and > 82% accuracy for detection of the S1-RBD antigen., Conclusion: The new S1-RBD antigen test is a rapid (15 min), sensitive, and specific assay that requires minimal sample preparation. Critically, the assay correlated closely with PCR-based methodology in nasopharyngeal swab samples, showing that the detected S1-RBD antigen levels correlate with SARS-CoV-2 virus load. Therefore, the new TRF lateral flow test for S1-RBD has potential application in point-of-care settings., (© 2022 The Authors. Journal of Clinical Laboratory Analysis published by Wiley Periodicals LLC.)

The effect of the chemical structure of selected phenolic acids on the molecular organization of gliadins was investigated with the application of Fourier Transform Infrared (FTIR) technique, steady-state, and time-resolved fluorescence spectroscopy. Hydroxybenzoic (4-hydroxybenzoic, protocatechuic, vanillic, and syringic) and hydroxycinnamic (coumaric, caffeic, ferulic, sinapic) acids have been used as gliadins modifiers. The results indicated that hydroxybenzoic acids due to their smaller size incorporate into spaces between two polypeptide chains and form a hydrogen bond with them leading to aggregation. Additionally, syringic acids could incorporate into hydrophobic pockets of protein. Whereas hydroxycinnamic acids, due to their higher stiffness and larger size, separated polypeptide chains leading to gliadin disaggregation. These acids did not incorporate into hydrophobic pockets.

C-peptide is a biomarker that has clinical implications for the diagnosis of a variety of diseases. In this study, an ultrasensitive time-resolved fluorescence lateral flow immunochromatographic assay (TRF-LFIA) method was established for the detection of C-peptides in human serum. The key to this method is the oriented immobilization of antibodies anti C-peptide on TRF microspheres that can sufficiently expose the antigen binding site. The limit of detection (LOD) of this method for C-peptide was 0.005 ng mL -1 , which is 10-fold less than that of TRF-LFIA method based on nonoriented immobilizing antibodies. The working range of this method was 0.005-250 ng mL -1 , and the spiked recoveries of C-peptide in human serum were 106.85%-116.40% with a CV value less than 10%. The test results of actual serum samples had good consistency (R 2  > 0.97) with the Roche Cobas 8000 automatic chemiluminescence immunoassay analyzer. This method can be utilized for the point-of-care testing (POCT) of C-peptide, and the oriented immobilizing method can also be used to construct highly sensitive probes to improve the sensitivity of other analytes in the POCT platform., (Copyright © 2022 Elsevier B.V. All rights reserved.)

To understand the photochemical behaviour of the polydopamine polymer in detail, one would also need to know the behaviour of its building blocks. The electronic absorption, as well as the fluorescence emission and excitation spectra of the dopamine were experimentally and theoretically investigated considering time-resolved fluorescence spectroscopy and first-principles quantum theory methods. The shape of the experimental absorption spectra obtained for different dopamine species with standard, zwitterionic, protonated, and deprotonated geometries was interpreted by considering the advanced equation-of-motion coupled-cluster theory of DLPNO-STEOM. Dynamical properties such as fluorescence lifetimes or quantum yield were also experimentally investigated and compared with theoretically predicted transition rates based on Fermi's Golden Rule-like equation. The results show that the photochemical behaviour of dopamine is strongly dependent on the concentration of dopamine, whereas in the case of a high concentration, the zwitterionic form significantly affects the shape of the spectrum. On the other hand, the solvent pH is also a determining factor for the absorption, but especially for the fluorescence spectrum, where at lower pH (5.5), the protonated and, at higher pH (8.0), the deprotonated forms influence the shape of the spectra. Quantum yield measurements showed that, besides the radiative deactivation mechanism characterized by a relatively small QY value, non-radiative deactivation channels are very important in the relaxation process of the electronic excited states of different dopamine species.

Thienoguanosine (thG) is an isomorphic fluorescent guanosine (G) surrogate, which almost perfectly mimics the natural G in DNA duplexes and may therefore be used to sensitively investigate for example protein-induced local conformational changes. To fully exploit the information given by the probe, we carefully re-investigated the thG spectroscopic properties in 12-bp duplexes, when the Set and Ring Associated (SRA) domain of UHRF1 flips its 5' flanking methylcytosine (mC). The SRA-induced flipping of mC was found to strongly increase the fluorescence intensity of thG, but this increase was much larger when thG was flanked in 3' by a C residue as compared to an A residue. Surprisingly, the quantum yield and fluorescence lifetime values of thG were nearly constant, regardless of the presence of SRA and the nature of the 3' flanking residue, suggesting that the differences in fluorescence intensities might be related to changes in absorption properties. We evidenced that thG lowest energy absorption band in the duplexes can be deconvoluted into two bands peaking at ∼350 nm and ∼310 nm, respectively red-shifted and blue-shifted, compared to the spectrum of thG monomer. Using quantum mechanical calculations, we attributed the former to a nearly pure ππ * excitation localized on thG and the latter to excited states with charge transfer character. The amplitude of thG red-shifted band strongly increased when its 3' flanking C residue was replaced by an A residue in the free duplex, or when its 5' flanking mC residue was flipped by SRA. As only the species associated with the red-shifted band were found to be emissive, the highly unusual finding of this work is that the brightness of thG in free duplexes as well as its changes on SRA-induced mC flipping almost entirely depend on the relative population and/or absorption coefficient of the red-shifted absorbing species., (© 2022 IOP Publishing Ltd.)