One-Pot Preparation of Ratiometric Fluorescent Molecularly Imprinted Polymer Nanosensor for Sensitive and Selective Detection of 2,4-Dichlorophenoxyacetic Acid.

The development of fluorescent molecular imprinting sensors for direct, rapid, and sensitive detection of small organic molecules in aqueous systems has always presented a significant challenge in the field of detection. In this study, we successfully prepared a hydrophilic colloidal molecular imprinted polymer (MIP) with 2,4-dichlorophenoxyacetic acid (2,4-D) using a one-pot approach that incorporated polyglycerol methacrylate (PGMMA-TTC), a hydrophilic macromolecular chain transfer agent, to mediate reversible addition-fragmentation chain transfer precipitation polymerization (RAFTPP). To simplify the polymerization process while achieving ratiometric fluorescence detection, red fluorescent CdTe quantum dots (QDs) and green fluorescent nitrobenzodiazole (NBD) were introduced as fluorophores (with NBD serving as an enhancer to the template and QDs being inert). This strategy effectively eliminated background noise and significantly improved detection accuracy. Uniform-sized MIP microspheres with high surface hydrophilicity and incorporated ratiometric fluorescent labels were successfully synthesized. In aqueous systems, the hydrophilic ratio fluorescent MIP exhibited a linear response range from 0 to 25 µM for the template molecule 2,4-D with a detection limit of 0.13 µM. These results demonstrate that the ratiometric fluorescent MIP possesses excellent recognition characteristics and selectivity towards 2,4-D, thus, making it suitable for selective detection of trace amounts of pesticide 2,4-D in aqueous systems.

Far-Red Fluorescent Proteins: Tools for Advancing In Vivo Imaging.

Far-red fluorescent proteins (FPs) have emerged as indispensable tools in in vivo imaging, playing a pivotal role in elucidating fundamental mechanisms and addressing application issues in biotechnology and biomedical fields. Their ability for deep penetration, coupled with reduced light scattering and absorption, robust resistance to autofluorescence, and diminished phototoxicity, has positioned far-red biosensors at the forefront of non-invasive visualization techniques for observing intracellular activities and intercellular behaviors. In this review, far-red FPs and their applications in living systems are mainly discussed. Firstly, various far-red FPs, characterized by emission peaks spanning from 600 nm to 650 nm, are introduced. This is followed by a detailed presentation of the fundamental principles enabling far-red biosensors to detect biomolecules and environmental changes. Furthermore, the review accentuates the superiority of far-red FPs in multi-color imaging. In addition, significant emphasis is placed on the value of far-red FPs in improving imaging resolution, highlighting their great contribution to the advancement of in vivo imaging.

EL4 Murine-Lymphoma-Stromal-Cell Fusion Hybrids Observed With Multiple Distinct Morphologies in the Primary Tumor and Metastatic Organs of a Syngeneic Mouse Model.

BACKGROUND/AIM: We and others have previously shown that cell fusion plays an important role in cancer metastasis. Color coding of cancer and stromal cells with spectrally-distinct fluorescent proteins is a powerful tool, as pioneered by our laboratory to detect cell fusion. We have previously reported color-coded cell fusion between cancer cells and stromal cells in metastatic sites by using color-coded EL4 murine lymphoma cells and host mice expressing spectrally-distinct fluorescent proteins. Cell fusion occurred between cancer cells or, between cancer cells and normal cells, such as macrophages, fibroblasts, and mesenchymal stem cells. In the present study, the aim was to morphologically classify the fusion-hybrid cells observed in the primary tumor and multiple metastases EL4 formed from cells expressing red fluorescent protein (RFP) in transgenic mice expressing green fluorescent protein (GFP), in a syngeneic model. MATERIALS AND METHODS: RFP-expressing EL4 murine lymphoma cells were cultured in vitro. EL4-RFP cells were harvested and injected intraperitoneally into immunocompetent transgenic C57/BL6-GFP mice to establish a syngeneic model. Two weeks later, mice were sacrificed and each organ was harvested, cultured, and observed using confocal microscopy. RESULTS: EL4 intraperitoneal tumors (primary) and metastases in the lung, liver, blood, and bone marrow were formed. All tumors were harvested and cultured. In all specimens, RFP-EL4 cells, GFP-stromal cells, and fused yellow-fluorescent hybrid cells were observed. The fused hybrid cells showed various morphologies. Immune cell-like round-shaped yellow-fluorescent fused cells had a tendency to decrease with time in liver metastases and circulating blood. In contrast fibroblast-like spindle-shaped yellow-fluorescent fused cells increased in the intraperitoneal primary tumor, lung metastases, and bone marrow. CONCLUSION: Cell fusion between EL4-RFP cells and GFP stromal cells occurred in primary tumors and all metastatic sites. The morphology of the fused hybrid cells varied in the primary and metastatic sites. The present results suggest that fused cancer and stromal hybrid cells of varying morphology may play an important role in cancer progression.

A novel red fluorescent and dynamic nanocomposite hydrogel based on chitosan and alginate doped with inclusion complex of carbon dots.

Red fluorescent hydrogels possessing injectable and self-healing properties have widespread potential in biomedical field. It is still a challenge to achieve a biomacromolecules based dynamic hydrogels simultaneously combining with excellent red fluorescence, good mechanical properties, and biocompatibility. Here we first explore hydrophilic inclusion complex of (R-CDs@α-CD) derived from hydrophobic red fluorescent carbon dots (R-CDs) and α-cyclodextrin (α-CD), and then achieved a red fluorescent and dynamic polysaccharide R-CDs@α-CD/CEC-l-OSA hydrogel. The nanocomposite hydrogel can be fabricated through controlled doping of red fluorescent R-CDs@α-CD into dynamic polymer networks, taking reversibly crosslinked N-carboxyethyl chitosan (CEC) and oxidized sodium alginate (OSA) as an example. The versatile red fluorescent hydrogel simultaneously combines the features of injection, biocompatibility, and augmented mechanical properties and self-healing behavior, especially in rapid self-recovery even after integration. The R-CDs@α-CD uniformly dispersed into dynamic hydrogel played the role of killing two birds with one stone, that is, endowing red emission of a hydrophilic fluorescent substance, and improving mechanical and self-healing properties as a dynamic nano-crosslinker, via forming hydrogen bonds as reversible crosslinkings. The novel red fluorescent and dynamic hydrogel based on polysaccharides is promising for using as biomaterials in biomedical field.

Method to Assess the Intracellular Fate and Bioavailability of Clusterin Using Live Cell Confocal Microscopy Imaging.

Clusterin, also known as apolipoprotein J, is an ATP-independent holdase chaperone protein. Clusterin is involved in various functions including protein quality control and lipid transport. Though clusterin is secreted upon stress, the intracellular fate of clusterin after a stress response is not well understood. The protocol described here utilizes clusterin tagged to fluorescent proteins like green fluorescent protein and red fluorescent protein to understand the intracellular fate of clusterin.

Fluorescently Tagged Verticillium dahliae to Understand the Infection Process on Cotton (Gossypium hirsutum) and Weed Plant Species.

Verticillium wilt is a soil-borne disease caused by distinct vegetative compatibility groups (VCG) of the fungus Verticillium dahliae. Defoliating (VCG 1A) and non-defoliating (VCG 2A) pathotypes of V. dahliae have contributed to yield losses of cotton production in Australia. To study the virulence and the infection process of V. dahliae on cotton, two isolates, one representing each VCG, have been transformed with fluorescent protein genes. The transformants maintained their ability to infect the host, and both strains were observed to move through the plant vasculature to induce wilt symptoms. Furthermore, virulence testing suggests that the cotton V. dahliae strains can endophytically colonise common weed plant species found in the Australian landscape, and that is contrasted by their ability to infect and colonise native tobacco plants. The fluorescently labelled strains of V. dahliae not only allowed us to gain a thorough understanding of the infection process but also provided a method to rapidly identify recovered isolates from host colonisation studies.

Red Fluorescent Aminoferrocene (Pro)Drugs for in Cellulo and in Vivo Imaging.

Red fluorescent dyes are usually charged, lipophilic molecules with relatively high molecular weight, which tend to localize in specific intracellular locations, e. g., a cyanine dye Cy5 is biased towards mitochondria. They are often used as markers of biomolecules including nucleic acids and proteins. Since the molecular weight of the dyes is much smaller than that of the biomolecules, the labelling has a negligible effect on the properties of the biomolecules. In contrast, conjugation of the dyes to low molecular weight (pro)drugs can dramatically alter their properties. For example, conjugates of Cy5 with lysosome-targeting aminoferrocenes accumulate in mitochondria and exhibit no intracellular effects characteristic for the parent (pro)drugs. Herein we tested several neutral and negatively charged dyes for labelling lysosome-targeting aminoferrocenes 7 and 8 as well as a non-targeted control 3. We found that a BODIPY derivative BDP-TR exhibits the desired unbiased properties: the conjugation does not disturb the intracellular localization of the (pro)drugs, their mode of action, and cancer cell specificity. We used the conjugates to clarify the mechanism of action of the aminoferrocenes. In particular, we identified new intermediates, explained why lysosome-targeting aminoferrocenes are more potent than their non-targeted counterparts, and evaluated their distribution in vivo.

Elucidating photocycle in large Stokes shift red fluorescent proteins: Focus on mKeima.

Large Stokes shift red fluorescent proteins (LSS-RFPs) are genetically encoded and exhibit a significant difference of a few hundreds of nanometers between their excitation and emission peak maxima (i.e., the Stokes shift). These LSS-RFPs (absorbing blue light and emitting red light) feature a unique photocycle responsible for their significant Stokes shift. The photocycle associated with this LSS characteristic in certain RFPs is quite perplexing, hinting at the complex nature of excited-state photophysics. This article provides a brief review on the fundamental mechanisms governing the photocycle of various LSS-RFPs, followed by a discussion on experimental results on mKeima emphasizing its relaxation pathways which garnered attention due to its >200 nm Stokes shift. Corroborating steady-state spectroscopy with computational studies, four different forms of chromophore of mKeima contributing to the cis-trans conformers of the neutral and anionic forms were identified in a recent study. Furthering these findings, in this account a detailed discussion on the photocycle of mKeima, which encompasses sequential excited-state isomerization, proton transfer, and subsequent structural reorganization involving three isomers, leading to an intriguing temperature and pH-dependent dual fluorescence, is explored using broadband femtosecond transient absorption spectroscopy.

Live Cell Imaging and Real-Time Monitoring of Nucleolus Morphology and Mitophagy with a Red Fluorescent and Photostable rRNA-Specific Probe in Human Cancer Cells.

rRNAs are prevalent in living organisms. They are produced in nucleolus and mitochondria and play essential cellular functions. In addition to the primary biofunction in protein synthesis, rRNAs have been recognized as the emerging signaling molecule and drug target for studies on nucleolus morphology, mitochondrial autophagy, and tumor cell malignancy. Currently, only a few rRNA-selective probes have been developed, and most of them encounter the drawbacks of low water solubility, poor nuclear membrane permeability, short emission wavelength, low stability against photobleaching, and high cytotoxicity. These unfavorable properties of rRNA probes limit their potential applications. In the present study, we reported a new rRNA-selective and near-infrared fluorescent turn-on probe, 4MPS-TO, capable of tracking rRNA in live human cancer cells. The real-time monitoring performance in nucleolus morphology and mitochondrial autophagy is demonstrated in HeLa cells. The probe shows great application potential for being used as a rRNA-selective, sensitive, and photostable imaging tool in chemical biology study and drug screening.

Improved fluorescence-based assay for rapid screening and evaluation of SARS-CoV-2 main protease inhibitors.

The outbreak of coronavirus disease 2019 (COVID-19) pandemic caused by the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is a global threat to human health. In parallel with vaccines, efficacious antivirals are urgently needed. SARS-CoV-2 main protease (Mpro) is an attractive drug target for antiviral development owing to its key roles in virus replication and host immune evasion. Due to the limitations of currently available methods, the development of novel high-throughput screening assays is of the highest importance for the discovery of Mpro inhibitors. In this study, we first developed an improved fluorescence-based assay for rapid screening of Mpro inhibitors from an anti-infection compound library using a versatile dimerization-dependent red fluorescent protein (ddRFP) biosensor. Utilizing this assay, we identified MG-101 as a competitive Mpro inhibitor in vitro. Moreover, our results revealed that ensitrelvir is a potent Mpro inhibitor, but baicalein, chloroquine, ebselen, echinatin, and silibinin are not. Therefore, this robust ddRFP assay provides a faithful avenue for rapid screening and evaluation of Mpro inhibitors to fight against COVID-19.

Methods for the detection of intracellular calcium in filamentous fungi.

Calcium (Ca2+), a critical secondary messenger, is also known as the molecule of life and death. The cell responds to a minute change in Ca2+ concentration and tightly maintains Ca2+ homeostasis. Therefore, determining the cell Ca2+ level is critical to understand Ca2+ distribution in the cell and various cell processes. Many techniques have been developed to measure Ca2+ in the cell. We review here different methods used to detect and measure Ca2+ in filamentous fungi. Ca2+-sensitive fluorescent chlortetracycline hydrochloride (CTC), Ca2+-selective microelectrode, Ca2+ isotopes, aequorins, and RGECOs are commonly used to measure the Ca2+ level in filamentous fungi. The use of CTC was one of the earliest methods, developed in 1988, to measure the Ca2+ gradient in the filamentous fungus Neurospora crassa. Subsequently, Ca2+-specific microelectrodes were developed later in the 1990s to identify Ca2+ ion flux variations, and to measure Ca2+ concentration. Another method for quantifying Ca2+ is by using radio-labeled Ca2+ as a tracer. The usage of 45Ca to measure Ca2+ in Saccharomyces cerevisiae was reported previously and the same methodology was also used to detect Ca2+ in N. crassa recently. Subsequently, genetically engineered Ca2+ indicators (GECIs) like aequorins and RGECOs have been developed as Ca2+ indicators to detect and visualize Ca2+ inside the cell. In this review, we summarize various methodologies used to detect and measure Ca2+ in filamentous fungi with their advantages and limitations. •Chlortetracycline (CTC) fluorescence assay is used for visualizing Ca2+ level, whereas microelectrodes technique is used to determine Ca2+ flux in the cell.•Radioactive 45Ca is useful for quantification of Ca2+ in the cellular compartments.•Genetically modified calcium indicators (GECIs) are used to study Ca2+ dynamics in the cell.

[Preparation and characterization of a fluorogenic ddRFP-M biosensor as a specific SARS-CoV-2 main protease substrate].

The conventional peptide substrates of SARS-CoV-2 main protease (Mpro) are frequently associated with high cost, unstable kinetics, and multistep synthesis. Hence, there is an urgent need to design affordable and stable Mpro substrates for pharmacological research. Herein, we designed a functional Mpro substrate based on a dimerization-dependent red fluorescent protein (ddRFP) for the evaluation of Mpro inhibitors in vitro. The codon-optimized DNA fragment encoding RFP-A1 domain, a polypeptide linker containing Mpro cleavage sequence (AVLQS), and the RFP-B1 domain was subcloned into the pET-28a vector. After transformation into Escherichia coli Rosetta(DE3) cells, the kanamycin resistant transformants were selected. Using a low temperature induction strategy, most of the target proteins (ddRFP-M) presented in the supernatant fractions were collected and purified by a HisTrapTM chelating column. Subsequently, the inhibition of Mpro by ensitrelvir and baicalein was assessed using ddRFP-M assay, and the biochemical properties of ddRFP-M substrate were analyzed. Our results showed that the fluorogenic substrate ddRFP-M was successfully prepared from E. coli cells, and this biosensor exhibited the expected specificity, sensitivity, and reliability. In conclusion, the production of the fluorogenic substrate ddRFP-M provides an expedient avenue for the assessment of Mpro inhibitors in vitro.

Development of an activatable far-red fluorescent probe for rapid visualization of hypochlorous acid in live cells and mice with neuroinflammation.

Recent investigations have suggested that abnormally elevated levels of HOCl may be tightly related to the severity of neuroinflammation. Although some successes have been achieved, fluorescent probes with far-red fluorescence emission and capable of detecting HOCl with high specificity in pure aqueous solution are still urgently needed. Herein, a responsive far-red fluorescent probe, DCI-H, has been constructed to monitor HOCl activity in vivo and in vitro. DCI-H could rapidly respond to HOCl within 120 s and had a low detection limit for HOCl of 1.5 nM. Importantly, physiologically common interfering species, except for HOCl, did not cause a change in the fluorescence intensity of DCI-HOCl at 655 nm. The results of confocal imaging demonstrated the ability of DCI-H to visualize endogenous HOCl produced by MPO-catalyzed H2O2/Cl- and LPS stimulation. With the assistance of DCI-H, upregulation of HOCl levels was observed in the mice model of LPS-induced neuroinflammation. Thus, we believed that DCI-H provided a valuable tool for HOCl detection and diagnosis of inflammation-related diseases.

A Coumarin-Hemicyanine Deep Red Dye with a Large Stokes Shift for the Fluorescence Detection and Naked-Eye Recognition of Cyanide.

In this study, we synthesized a coumarin-hemicyanine-based deep red fluorescent dye that exhibits an intramolecular charge transfer (ICT). The probe had a large Stokes shift of 287 nm and a large molar absorption coefficient (ε = 7.5 × 105 L·mol-1·cm-1) and is best described as a deep red luminescent fluorescent probe with λem = 667 nm. The color of probe W changed significantly when it encountered cyanide ions (CN-). The absorption peak (585 nm) decreased gradually, and the absorption peak (428 nm) increased gradually, so that cyanide (CN-) could be identified by the naked eye. Moreover, an obvious fluorescence change was evident before and after the reaction under irradiation using 365 nm UV light. The maximum emission peak (667 nm) decreased gradually, whilst the emission peak (495 nm) increased gradually, which allowed for the proportional fluorescence detection of cyanide (CN-). Using fluorescence spectrometry, the fluorescent probe W could linearly detect CN- over the concentration range of 1-9 µM (R2 = 9913, RSD = 0.534) with a detection limit of 0.24 µM. Using UV-Vis spectrophotometry, the linear detection range for CN- was found to be 1-27 µM (R2 = 0.99583, RSD = 0.675) with a detection limit of 0.13 µM. The sensing mechanism was confirmed by 1H NMR spectroscopic titrations, 13C NMR spectroscopy, X-ray crystallographic analysis and HRMS. The recognition and detection of CN- by probe W was characterized by a rapid response, high selectivity, and high sensitivity. Therefore, this probe provides a convenient, effective and economical method for synthesizing and detecting cyanide efficiently and sensitively.

Experimental genetic crosses in tsetse flies of the livestock pathogen Trypanosoma congolense savannah.

BACKGROUND: In tropical Africa animal trypanosomiasis is a disease that has severe impacts on the health and productivity of livestock in tsetse fly-infested regions. Trypanosoma congolense savannah (TCS) is one of the main causative agents and is widely distributed across the sub-Saharan tsetse belt. Population genetics analysis has shown that TCS is genetically heterogeneous and there is evidence for genetic exchange, but to date Trypanosoma brucei is the only tsetse-transmitted trypanosome with experimentally proven capability to undergo sexual reproduction, with meiosis and production of haploid gametes. In T. brucei sex occurs in the fly salivary glands, so by analogy, sex in TCS should occur in the proboscis, where the corresponding portion of the developmental cycle takes place. Here we test this prediction using genetically modified red and green fluorescent clones of TCS. METHODS: Three fly-transmissible strains of TCS were transfected with genes for red or green fluorescent protein, linked to a gene for resistance to the antibiotic hygromycin, and experimental crosses were set up by co-transmitting red and green fluorescent lines in different combinations via tsetse flies, Glossina pallidipes. To test whether sex occurred in vitro, co-cultures of attached epimastigotes of one red and one green fluorescent TCS strain were set up and sampled at intervals for 28 days. RESULTS: All interclonal crosses of genetically modified trypanosomes produced hybrids containing both red and green fluorescent proteins, but yellow fluorescent hybrids were only present among trypanosomes from the fly proboscis, not from the midgut or proventriculus. It was not possible to identify the precise life cycle stage that undergoes mating, but it is probably attached epimastigotes in the food canal of the proboscis. Yellow hybrids were seen as early as 14 days post-infection. One intraclonal cross in tsetse and in vitro co-cultures of epimastigotes also produced yellow hybrids in small numbers. The hybrid nature of the yellow fluorescent trypanosomes observed was not confirmed by genetic analysis. CONCLUSIONS: Despite absence of genetic characterisation of hybrid trypanosomes, the fact that these were produced only in the proboscis and in several independent crosses suggests that they are products of mating rather than cell fusion. The three-way strain compatibility observed is similar to that demonstrated previously for T. brucei, indicating that a simple two mating type system does not apply for either trypanosome species.

Non-invasive Fluorescence Imaging of Breast Cancer Metastasis to the Brain in an Orthotopic Nude-mouse Model With Very-narrow-band-width Laser Excitation of Red Fluorescent Protein Resulting in an Ultra-bright Signal Without Skin Autofluorescence.

BACKGROUND/AIM: Breast-cancer metastasis to the brain is an intractable disease. To discover improved therapy for this disease, we developed a precise non-invasively-imageable orthotopic nude-mouse model, using very-narrow-band-width laser fluorescence excitation. MATERIALS AND METHODS: Female nu/nu nude mice, aged 4-8 weeks, were inoculated through the midline of the skull with triple-negative human MDA-MB-231 breast cancer cells (5×105) expressing red fluorescent protein (RFP). The mice were imaged with the Analytik Jena UVP Biospectrum Advanced at 520 nm excitation with peak emission at 605 nm. RESULTS: Three weeks after injection of MDA-MB-231-RFP cells in the brain, non-invasive fluorescence images of the breast tumor growing on the brain were obtained. The images of the tumor were very bright, with well-defined margins with no detectable skin autofluorescence background. Images obtained at various angles showed that the extent of the tumor margins could be precisely measured. A skin flap over the skull confirmed that the tumor was growing on the surface of the brain which is a frequent occurrence in breast cancer. CONCLUSION: A precise orthotopic model of RFP-expressing breast-cancer metastasis to the brain was developed that could be non-invasively imaged with very-narrow-band-width laser excitation, resulting in an ultra-bright, ultra-low-background signal. The model will be useful in discovering improved therapeutics for this recalcitrant disease.

Targeting Ultrafast Spectroscopic Insights into Red Fluorescent Proteins.

Red fluorescent proteins (RFPs) represent an increasingly popular class of genetically encodable bioprobes and biomarkers that can advance next-generation breakthroughs across the imaging and life sciences. Since the rational design of RFPs with improved functions or enhanced versatility requires a mechanistic understanding of their working mechanisms, while fluorescence is intrinsically an ultrafast event, a suitable toolset involving steady-state and time-resolved spectroscopic techniques has become powerful in delineating key structural features and dynamic steps which govern irreversible photoconverting or reversible photoswitching RFPs, and large Stokes shift (LSS)RFPs. The pertinent cis-trans isomerization and protonation state change of RFP chromophores in their local environments, involving key residues in protein matrices, lead to rich and complicated spectral features across multiple timescales. In particular, ultrafast excited-state proton transfer in various LSSRFPs showcases the resolving power of wavelength-tunable femtosecond stimulated Raman spectroscopy (FSRS) in mapping a photocycle with crucial knowledge about the red-emitting species. Moreover, recent progress in noncanonical RFPs with a site-specifically modified chromophore provides an appealing route for efficient engineering of redder and brighter RFPs, highly desirable for bioimaging. Such an effective feedback loop involving physical chemists, protein engineers, and biomedical microscopists will enable future successes to expand fundamental knowledge and improve human health.

Backbone 1H, 15N and 13C resonance assignments of the 27kDa fluorescent protein mCherry.

mCherry is one of the most successfully applied monomeric red fluorescent proteins (RFPs) for in vivo and in vitro imaging. However, questions pertaining to the photostability of the RFPs remain and rational further engineering of their photostability requires information about the fluorescence quenching mechanism in solution. To this end, NMR spectroscopic investigations might be helpful, and we present the near-complete backbone NMR chemical shift assignment to aid in this pursuit.

Structural Characterization of Fluorescent Proteins Using Tunable Femtosecond Stimulated Raman Spectroscopy.

The versatile functions of fluorescent proteins (FPs) as fluorescence biomarkers depend on their intrinsic chromophores interacting with the protein environment. Besides X-ray crystallography, vibrational spectroscopy represents a highly valuable tool for characterizing the chromophore structure and revealing the roles of chromophore-environment interactions. In this work, we aim to benchmark the ground-state vibrational signatures of a series of FPs with emission colors spanning from green, yellow, orange, to red, as well as the solvated model chromophores for some of these FPs, using wavelength-tunable femtosecond stimulated Raman spectroscopy (FSRS) in conjunction with quantum calculations. We systematically analyzed and discussed four factors underlying the vibrational properties of FP chromophores: sidechain structure, conjugation structure, chromophore conformation, and the protein environment. A prominent bond-stretching mode characteristic of the quinoidal resonance structure is found to be conserved in most FPs and model chromophores investigated, which can be used as a vibrational marker to interpret chromophore-environment interactions and structural effects on the electronic properties of the chromophore. The fundamental insights gained for these light-sensing units (e.g., protein active sites) substantiate the unique and powerful capability of wavelength-tunable FSRS in delineating FP chromophore properties with high sensitivity and resolution in solution and protein matrices. The comprehensive characterization for various FPs across a colorful palette could also serve as a solid foundation for future spectroscopic studies and the rational engineering of FPs with diverse and improved functions.

Functional Group Modulation in Carbon Quantum Dots for Accelerating Photocatalytic CO2 Reduction.

This study investigates the mechanism behind the enhanced photocatalytic performance of carbon quantum dot (CQD)-induced photocatalysts. Red luminescent CQDs (R-CQDs) were synthesized using a microwave ultrafast synthesis strategy, exhibiting similar optical and structural properties but varying in surface functional group sites. Model photocatalysts were synthesized by combining R-CQDs with graphitic carbon nitride (CN) using a facile coupling technique, and the effects of different functionalized R-CQDs on CO2 reduction were investigated. This coupling technique narrowed the band gap of R1-CQDs/CN, made the conduction band potentials more negative, and made photogenerated electrons and holes less likely to recombine. These improvements greatly enhanced the deoxygenation ability of the photoinduced carriers, increased light absorption of solar energy, and raised the carrier concentration, resulting in excellent stability and remarkable CO production. R1-CQDs/CN demonstrated the highest photocatalytic activity, with CO production up to 77 µmol g-1 within 4 h, which is approximately 5.26 times higher than that of pure CN. Our results suggest that the superior photocatalytic performance of R1-CQDs/CN arises from its strong internal electric field and high Lewis acidity and alkalinity, attributed to the abundant pyrrolic-N and oxygen-containing surface groups, respectively. These findings offer a promising strategy for producing efficient and sustainable CQD-based photocatalysts to address global energy and environmental problems.