Fluorescence Response Research Articles

Variable bioenergetic sensitivity of neurons and astrocytes to insulin and extracellular glucose

Energy flow within cellular elements of the brain is a well-orchestrated, tightly regulated process, however, details underlying these functions at the single-cell level are still poorly understood. Studying hypometabolism in aging and neurodegenerative diseases may benefit from experimentation on unicellular bioenergetics. Here, we examined energy status in neurons and astrocytes using mixed hippocampal cultures and PercevalHR, an ATP:ADP nanosensor. We assessed exposures of several compounds including KCl, glutamate, FCCP, insulin, and glucose. A mitochondrial stress test was performed, and PercevalHR’s fluorescence was corrected for pH using pHrodo. Results demonstrate that PercevalHR can reliably report on the energetic status of two cell types that communicate in a mixed-culture setting. While KCl, glutamate, and FCCP showed clear changes in PercevalHR fluorescence, insulin and glucose responses were found to be more subtle and sensitive to extracellular glucose. These results may highlight mechanisms that mediate insulin sensitivity in the brain.

Application of RET Approach for Ratiometric Response Enhancement of ICT Fluorescent Hg2+ Probe based on Crown-containing Styrylpyridinium Dye.

Styrylpyridinium dye bearing azadithia-15-crown-5 ether receptor group SP and 4-alkoxy-1,8-naphthalimide fluorophore were linked using copper-catalyzed azide-alkyne cycloaddition click reaction to afford dyad compound NI-SP. Chemosensor NI-SP exhibited selective ratiometric fluorescent response to the presence of Hg2+ in aqueous solution due to the interplay between resonance energy transfer (RET) and intramolecular charge transfer (ICT) processes occurred upon excitation. The observed switching of the ratio of emission intensities in the blue and red channels R was higher than in the case of monochromophoric styrylpyridine derivative SP showing ratiometric response based on ICT mechanism only. Biological studies revealed that NI-SP penetrates into human lung adenocarcinoma A549 cells and accumulates in cytoplasm and lysosomes. When cells were pre-incubated with mercury (II) perchlorate, the ratio R was increased 2.6 times, which enables detection of intracellular Hg2+ ions and their quantitative analysis in the 0.7-6.0 μM concentration range.

Dimensionality and Molecular Packing Control of Covalent Organic Frameworks through Pendant Group Design.

Tuning the dimensions and molecular packing geometry of crystalline organic frameworks and polymers represents an important challenge for reticular chemistry. Here we show that for extended structures made of 1,3,6,8-tetrakis(4-aminophenyl)pyrene (PyTTA) linked with methoxy group functionalized terephthalaldehyde aldehydes, simple substituents on the aldehyde linker can have profound structure directing effects due to noncovalent interactions. Specifically, reacting 2,3-dimethoxyterephthalaldehyde with PyTTA gives a 2D covalent organic framework with unique AA-inclined-AA stacking and bilayer pyrene motifs, whereas reacting 2,5-dimethoxyterephthalaldehyde with PyTTA gives a 1D crystalline polymer with AB stacking and isolated pyrene motifs. Both materials show high crystallinity, allowing for atomic resolution structure determination using three-dimensional electron diffraction, and the similarity of their fluorescence properties shows the electronic structures of pyrene-based extended structures mostly depends on the in-plane structures, which is supported by density functional theory calculations. These two pyrene-based extended structures also show different fluorescence responses to organic vapors due to different pore environments. The current work shows the potential of noncovalent interactions in the reticular design of functional organic materials.

Bidirectional "Win-Win": Asymmetrical Nanobowl-Coupled Aggregation-Induced Emissive Nanosilicon-Enhanced Immunochromatographic Strips for the Ultrasensitive Detection of Salmonella typhimurium.

The lack of nanoprobes with an efficient signal response and overlook of cooperation between nanoprobes can be responsible for the unsatisfactory analytical performance of immunochromatographic strips (ITSs). Herein, asymmetrical nanobowl-confined innumerable gold nanoparticles (AuNPs) (AuNPs@AFRNBs) to enhance the light absorption are developed for quenching the fluorescence of aggregation-induced emissive (AIE) nanosilicons, which is used for the construction of a bidirectional complementary-enhanced ITS (BC-ITS) to ultrasensitively detect Salmonella typhimurium (S. typhimurium). Briefly, density functional theory-screened AIEgens with highly fluorescent brightness are confined in nanosilicons, and the nanoconfinement has improved the fluorescent brightness by 6.78-fold compared to the free AIEgens. Moreover, the substituent group effect has also enhanced the fluorescence of the prepared fluorescent nanosilicon by 10,000-fold in ITSs. By virtue of the superior light absorption of AuNPs@AFRNBs, the BC-ITS exhibits a bidirectional "win-win" performance for the sensitive monitoring of S. typhimurium: a "turn-on" mode with a high-brightness colorimetric response and an inverse "turn-off" fluorescence response, whose limits of detection are 364 and 302 CFU mL-1, respectively, which is approximately 100-fold more sensitive than the traditional AuNPs-ITS. Furthermore, the BC-ITS can be successfully used to identify S. typhimurium in milk, illustrating the superiority of the developed BC-ITS in point-of-care diagnosis.

Design and synthesis of naphthalimide-based CO fluorescent probes realizing tricolor detection and turn-on/off response

Carbon monoxide (CO) fluorescent probes have attracted tremendous attention attributed to their low detection limit, high specificity, simple operation and good biocompatibility. However, the relationship between the molecular structure and the fluorescence response signal still needs to be systematically elucidated for different applications. Herein, three different CO fluorescent probes based on naphthalimide derivative with blue emission (B-NIA), green emission (G-NIA), and near-infrared emission (R-NIA) were designed and synthesized. Three-primary colors and opposite turn-on or turn-off fluorescent responses to CO were achieved by regulating the conjugation system of fluorophores and the position of fluorescent recognition group. The change of fluorescence response and sensing mechanism for CO detection was studied by theory calculation and mass spectrometry analysis. All the B/G/R-NIA possessed high sensitivity for CO detection. Notably, the R-NIA emerged with a noticeable NIR fluorescence response to CO with a low detection limit (0.61 µM) and high selectivity and relative pH stability. In addition, the R-NIA showed particularly low cytotoxicity and has been successfully used to detect CO in living cells. These studies provided the theoretical reference and technical route to synthesize fluorescent probes with different emission wavelengths and opposite fluorescent responses to CO for various application scenarios

Novel quinoline-based chemosensor as specific fluorescence sensing of copper ions in cancer cells and for organelle-specific imaging application

This work reports a simple synthesis of a quinoline-based organic molecule, (E)-N′-((6-methoxy-2-oxo-1,2-dihydroquinolin-3-yl)methylene) picolinohydrazide (PHMQ), and its ability to distinguish Cu2+ ion. Through the use of spectroscopic and photometric titration techniques, PHMQ’s selectivity toward various cations and anions was examined. In just two minutes, PHMQ showed fast detection of Cu2+ ions, and clear spectral fluctuations proposed persistent PHMQ-Cu2+ complex formation. The calculated emission quenching constant (1.46 × 105 L/mol) and binding constant (1.25 × 104 L/mol) values suggested that strong emission quenching occurs between the PHMQ and Cu2+ ions. A linear reduction in fluorescence response was noted for Cu2+ ions concentrations between 2–26 μM, with a detection limit of 43.4 nM. Job’s plot, ESI-mass, NMR titration experiments, and density functional theory (DFT) simulations established a 1:1 stoichiometry for the PHMQ-Cu2+ complex. Additionally, PHMQ was efficaciously utilized to quantify Cu2+ ions in drinking and tap water samples and HeLa cells, illustrating durable cellular penetrability and a fluorescence ’Turn-Off’ response to Cu2+ ions. This sensor also established usefulness in mitochondrial-targeted imaging. For the detection of Cu2+ ions in biological and environmental environments, PHMQ is a promising detector due to its high sensitivity, selectivity, and quick response.

A Highly Selective and Sensitive Carbazole-Based Fluorescent Probe for Peroxynitrite Detection and Cellular Imaging

Peroxynitrite, a colorless and odorless reactive nitrogen species, plays a critical role in various physiological processes within biological systems. Therefore, developing a reliable method for quantitative detection of peroxynitrite in living systems is essential. In this study, we synthesized a simple, two-step, reaction-based fluorescent probe, designated as EHMC, specifically for peroxynitrite detection. The synthesis involved the use of 4-(9-phenyl-9H-carbazol-3-yl)benzaldehyde as the fluorophore and 2-hydrazineylpyridine as the recognition group, both of which exhibit inherently weak fluorescence. Upon oxidation of the C=N bond by peroxynitrite, EHMC undergoes conversion into an aldehyde, resulting in a significant fluorescence response. EHMC demonstrated high selectivity and sensitivity, achieving a detection limit of 5.43 µM under physiological conditions. Additionally, EHMC was successfully applied to HeLa cellular models, where it exhibited excellent cellular imaging capability for peroxynitrite detection in living systems.

A simple and enzyme-free method for sensitive p53 analysis based on DNAzyme-mediated signal amplification

There is an urgent demand for a simple yet extremely accurate biosensor to analyze tumorigenesis. Herein, we present a novel fluorescent and enzyme-free approach for detecting p53 gene cascading proximity ligation-mediated catalytic hairpin assembly and DNAzyme-assisted signal reaction. When the target p53 gene is present, the interaction between p53 and L1 and L2 chains initiates catalytic hairpin assembly and subsequently exposes DNAzyme in the P3 probe. The exposed DNAzyme binds with the loop region of the P4 probe and generates a nicking site, resulting in the release of a significant amount of ATMND that is conjugated in the stem section of P4. This leads to an amplified fluorescence response, which serves as a fluorescence signal for the detection of the p53 gene. This method allows for the accurate and sensitive identification of the p53 gene, exhibiting a linear reaction range of 1 fM to 1 nM, with a limit of detection as low as 0.23 fM. Furthermore, this fluorescent method has been utilized for the examination of clinical samples with a favorable recovery rate. Crucially, this versatile platform may be expanded to analyze different targets by changing the corresponding recognition unit, showing great potential for point-of-care testing in tumorigenesis analysis.

BODIPY-derived fluorescent probes for targeting and tracking lipid droplets dynamics

Lipid droplets (LDs) are ubiquitous intracellular organelles present in various cell types, ranging from adipocytes to cancer cells. Despite their involvement in numerous diseases, current methodologies for monitoring LD dynamics rely primarily on static imaging with a limited selection of fluorescent dyes. In this study, we introduce two novel BODIPY-derived fluorophores designed to facilitate real-time monitoring of LD dynamics. By incorporating triphenylamino (TPA) moieties into the BODIPY core both asymmetrically and symmetrically, we modulated the fluorescence response to the green (GFP) and deep-red (Cy5) confocal channels, respectively, thus allowing dual-channel fluorescence capability for high-resolution, dynamic imaging of LDs within live cells, providing new insights into their behavior.

4,4'-Sulfonyldianiline Derived Aggregation-Induced Emission Luminogen for the Detection of Ofloxacin.

The excessive use of antibiotic ofloxacin (Oflx) can cause serious detrimental effects to human health. Therefore, the utmost research priority is required to develop facile methods to detect Oflx. Herein, a V-shaped aggregation-induced emission (AIE) active Schiff base SDANA was introduced for the fluorescent turn-on detection of Oflx. The Schiff base SDANA was synthesized by condensing 4,4'-sulfonyldianiline with two equivalents of 2-hydroxy-1-naphthaldehyde. The nearly non-fluorescent SDANA in DMSO showed strong orange emission with the increase in HEPES buffer (H2O, 10 mM, pH 7.4) fractions in DMSO from 70 %-95 % due to the combined effects of AIE and ESIPT. The DLS and SEM analyses were performed to complement the formation of self-aggregates of SDANA. With the addition of Oflx, the fluorescence emission of AIE luminogen (AIEgen) SDANA (λem=575 nm, λex=400 nm) was blue-shifted and enhanced at 530 nm. The interactions of Oflx over the surface of SDANA aggregates disrupted the intramolecular charge transfer and aggregation morphology of SDANA, which gave a distinct fluorescence response to detect Oflx. The detection limit for Oflx was estimated as 0.81 μM, and the developed probe AIEgen SDANA was applied for the quantification of Oflx in human blood serum.

A handheld fluorescent platform integrated with a Sm(III)-CdTe quantum dot-based ratiometric nanoprobe for point-of-use determination of phosphate.

Phosphate (Pi) is crucial for various physiological processes and aquatic environments, which emphasizes the need for a simple, on-site sensor to promptly detect Pi for human health and environmental conservation. In this study, we propose a dual-emission ratiometric fluorescence sensor for highly sensitive and visual Pi detection. The sensor employs samarium ions (Sm3+) as a core component, with cadmium telluride quantum dots (CdTe QDs) and ofloxacin (OFL) serving as signal carriers. The CdTe-Sm(III)-OFL nanoprobe emits a purple fluorescence resulting from the red fluorescence of CdTe QDs and the blue-green fluorescence of OFL. The fluorescence of OFL is quenched by Sm3+ through fluorescence resonance energy transfer (FRET). Upon Pi interaction, the FRET process is disrupted due to the competitive Pi-Sm3+ binding, which leads to the fluorescence recovery of OFL while the red fluorescence of CdTe remains steady. This enables the construction of a ratiometric fluorescent sensor for Pi detection, manifesting as a color change from purple to blue. The sensor demonstrated a linear response for Pi detection within the range of 0.1-75 μM, with a low detection limit of 17.0 nM. By utilizing the distinct fluorescence responses of various physiological phosphates and employing chemometrics, this innovative dual-emission sensor accurately distinguishes among different physiological phosphates. Furthermore, a portable lab-on-paper device based on CdTe-Sm(III)-OFL, coupled with a smartphone-integrated mini-device, is developed for swift Pi detection using an ordinary smartphone. Analytical performance validated on environmental and biological samples demonstrates the sensor's excellent robustness and adaptability. This study introduces a pioneering approach to fabricate ratiometric fluorescence sensors and customize portable, cost-effective mini-devices for precise target detection, thus opening avenues for advanced sensing strategies in various applications.

Determination and analysis of spectral fluorescence cross-sections of bacteria aerosols

Fluorescence cross-sections of single bacteria provide information on the strength of fluorescence response of bacteria for particular excitation conditions and give hints about a possible distinguishability of bacteria by fluorescence techniques. In this report, the fluorescence cross-sections of dry bacteria aerosols are derived from fluorescence spectra by absolute calibration of the measuring system. The fluorescence is measured in a continuous aerosol flow. The aerosol concentration is calculated from microscopic analyses of bacteria samples taken from the aerosol flow. Established calibration methods such as Mie scattering from monodisperse spheres and Raman scattering from water are applied to the same set of fluorescence spectra. The cross-section data obtained are evaluated with regard to their independency from the applied measuring system. The results are compared to data published in the literature. Polarization effects are identified to contribute to the discrepancies between the different datasets.Graphical abstract

A promising platform for the rapid and sensitive sensing of mercury ion and its applications in biological and environmental system

Mercury ion (Hg2+) is one of harmful heavy metal ions that can accumulate inside the human organism and cause some health problems. Herein, a novel NBD derivative, 1-(2-((7-nitrobenzo[c] [1,2,5] oxadiazol-4-yl) amino) ethyl)-3-phenylthiourea (NBD-SCN), has been reasonably designed, which was simply synthesized by introducing NBD moiety as the fluorophore, phenyl-isothionitrate as the reaction site and ethylenediamine as a linker group, resulting in intense fluorescence emission with high fluorescence quantum yields. Intriguingly, probe NBD-SCN provides a dual-mode colorimetric and fluorescence response pattern for the sensing of mercury ion with great limit of detections (3.6 nM) and rapid response time (4 s). The strong combination of mercury ions and sulfur atoms leads to intramolecular cyclization, causing the color of probe solution changed from orange to pale yellow as well as a complete vanishing of fluorescence intensity. The proposed reaction mechanism was verified by fluorescence and UV titration, 1H NMR, FTIR, HRMS analyses and DFT studies. Motivated by an impressive chromatic variation, test strip sensing platform are facilely constructed for real-time and on-site measurement of Hg2+ levels. Furthermore, confocal imaging experiments verify that this probe can be used for monitoring mercury ion in living mung bean sprouts and in biological systems in real time.

Genetically encoded green fluorescent sensor for probing sulfate transport activity of solute carrier family 26 member a2 (Slc26a2) protein

Genetically encoded fluorescent biosensors became indispensable tools for biological research, enabling real-time observation of physiological processes in live cells. Recent protein engineering efforts have resulted in the generation of a large variety of fluorescent biosensors for a wide range of biologically relevant processes, from small ions to enzymatic activity and signaling pathways. However, biosensors for imaging sulfate ions, the fourth most abundant physiological anion, in mammalian cells are still lacking. Here, we report the development and characterization of a green fluorescent biosensor for sulfate named Thyone. Thyone, derived through structure-guided design from bright green fluorescent protein mNeonGreen, exhibited a large negative fluorescence response upon subsecond association with sulfate anion with an affinity of 11 mM in mammalian cells. By integrating mutagenesis analyses with molecular dynamics simulations, we elucidated the molecular mechanism of sulfate binding and revealed key amino acid residues responsible for sulfate sensitivity. High anion selectivity and sensitivity of Thyone allowed for imaging of sulfate anion transients mediated by sulfate transporter heterologously expressed in cultured mammalian cells. We believe that Thyone will find a broad application for assaying the sulfate transport in mammalian cells via anion transporters and exchangers.

Synthesis of BODIPY-pyrrolo[3,4-b]pyridin-5-ones via Ugi-Zhu/cascade reactions and studies of fluorescence response toward viscosity.

A series of seven new meso-phenyl BODIPY-pyrrolo[3,4-b]pyridin-5-one conjugates were synthesized in one experimental step by using a Sc(III)-catalyzed Ugi-Zhu three-component reaction coupled to a cascade sequence (aza Diels-Alder/N-acylation/aromatization) as post-MCR functionalization process. Further experimental studies were performed behind understanding the fluorescence response toward viscosity. All compounds exhibited a linear response between increasing viscosity (DMSO and glycerol mixtures) and fluorescence intensity. The different substituents also influenced the photophysical properties. Furthermore, in DMSO all compounds exhibited dual emission. Each band is attributed to the pyrrolo[3,4-b]pyridin-5-one and BODIPY moieties, respectively. The electronic structure of all compounds was computed by DFT and TD-DFT calculations, allowing to determine the molecular orbitals involved in the electronic transitions.

Chemical Capture and Sensing of Bovine Serum Albumin with Phenothiazinylcarbaldehyde-Labeled 2′-Deoxyuridine

AbstractChemical-capture-mediated sensing has had a great impact on proteomic research. Toward this end, we demonstrate the chemical trapping of BSA by the reactive formyl functionality of a newly developed fluorescent nucleoside probe, formylphenothiazine-labeled-2′-deoxyuridine. The probe is capable of trapping BSA via Schiff base formation leading to fluorescence ‘switch-on’ sensing with a large hypsochromic shift of ca. 100 nm. The α-amylase does not show any significant change in fluorescence response, demonstrating the efficiency of the probe in selective sensing of BSA. Docking studies suggest the preferential interaction of the phenothiazinylcarbaldehyde-labeled dU with the residual amino acids in site I of the BSA protein as compared to site II.

Photochemical and electrochemical behavior of a molecular probe: Fluorescence on-off-on response to detect multiple ions (Cu2+, ClO− and CN−) with different rate of reaction

A molecular probe 3 containing phenyl substituted imidazolyl unit and diaminomaleonitrile (DAMN) moiety has been synthesized and characterized. Photophysical and electrochemical behavior probe 3 were studied in partial aqueous medium (ACN/H2O, 9.5:0.5 v/v). Probe 3 upon interaction with different class of ions showed sensitivity for Cu2+, ClO− and CN− ions with different response time and reaction rates due to variation in intramolecular charge transfer (ICT) process. Both Cu2+ and ClO− ions displayed enhanced emission due to hydrolysis of imine bond to form compound 2 in the medium whereas, with CN− fluorescence quenching was observed due to the deprotonation of –OH/–NH2 functions. The kinetic and optical behavior of the probe suggested that interaction occurred in two steps with Cu2+ (complexation and hydrolysis; absk1,2,3(Cu2+) = 1.17, 0.74 and 0.06 s−1) and ClO− (hydrolysis and deprotonation; absk1,2(ClO−) = 3.0 and 0.60 s−1) ions while CN− (absk1,2(CN−) = 2.13 and 1.66 s−1) induces deprotonation in one step. The interaction of probe 3 with ClO− was relatively fast than with Cu2+/CN− ions. Job's plot analysis between probe 3 and ions revealed a 1:1 (Cu2+/ClO−) and 1:2 (CN−) ratio stoichiometry with good limit of detection (nM) and binding affinity (LOD, 17; KCu2+ = 3.23 × 106 M−1; LOD, 18.5; KClO− = 5.23 × 105 M−1 and LOD, 23.2; KCN− = 5.50 × 109 M−2) respectively. Probe 3 displayed a naked–eye sensitive fluorescence response to detect tested ions on test paper strips and good recovery percentage of ions in real water sample analysis.

Seasonal biases in fluorescence-estimated chlorophyll-a derived from biogeochemical profiling floats

Marine phytoplankton biomass and chlorophyll-a concentration are often estimated from pigment fluorescence measurements, which have become routine despite known variability in the fluorescent response for a given amount of chlorophyll-a. Here, we present a near-global, monthly climatology of chlorophyll-a fluorescence measurements from profiling floats combined with ocean color satellite estimates of chlorophyll-a concentration to illuminate seasonal biases in the fluorescent response and expand upon previously observed regional patterns in this bias. Global biases span over an order of magnitude, and can vary seasonally by a factor of 10. An independent estimate of chlorophyll-a from light attenuation shows similar global patterns in the chlorophyll-fluorescence bias when compared to biases derived from satellite estimates. Without accounting for these biases, studies or models using fluorescence-estimated chlorophyll-a will inherit the seasonal and regional biases described here.

Synthesis and characterization of core–shell magnetic molecularly imprinted polymer nanocomposites for the detection of interleukin-6

Interleukin-6 (IL-6) belongs to the cytokine family and plays a vital role in regulating immune response, bone maintenance, body temperature adjustment, and cell growth. The overexpression of IL-6 can indicate various health complications, such as anastomotic leakage, cancer, and chronic diseases. Therefore, the availability of highly sensitive and specific biosensing platforms for IL-6 detection is critical. In this study, for the first time, epitope-mediated IL-6-specific magnetic molecularly imprinted core–shell structures with fluorescent properties were synthesized using a three-step protocol, namely, magnetic nanoparticle functionalization, polymerization, and template removal following thorough optimization studies. The magnetic molecularly imprinted polymers (MMIPs) were characterized using dynamic and electrophoretic light scattering (DLS and ELS), revealing a hydrodynamic size of 169.9 nm and zeta potential of +17.1 mV, while Fourier transform infrared (FTIR) spectroscopy and fluorescence spectroscopy techniques showed characteristic peaks of the polymer and fluorescent tag, respectively. Scanning electron microscopy (SEM) and high-resolution transmission electron microscopy (HRTEM) investigations confirmed the successful encapsulation of the magnetic core within the ca. 5-nm-thick polymeric shell. The MMIP-based electrochemical sensing platform achieved a limit of detection of 0.38 pM within a linear detection range of 0.38–380 pM, indicating high affinity (dissociation constant KD = 1.6 pM) for IL-6 protein in 50% diluted serum samples. Moreover, comparative investigations with the non-imprinted control polymer demonstrated an imprinting factor of 4, confirming high selectivity. With multifunctional features, including fluorescence, magnetic properties, and target responsiveness, the synthesized MMIPs hold significant potential for application in various sensor techniques as well as imaging.

An Acid-Activatable Fluorescent Probe for Sulfur Dioxide in Traditional Chinese Medicines and Living Cells.

Excessive sulfur dioxide (SO₂) disturbs physiology of lysosomes causing diseases and threatening human health. A fluorescent probe has been regarded as one of the most attractive approaches, which is compatible with living cells and possesses high sensitivity. However, most of fluorescent probes' reaction sites are activated before they reach the destination. In this work, an acid-activatable fluorescent probe PT1 was synthesized, characterized, and used for SO2 detection. The introduction of oxazolines in PT1 enables the intelligent response of probe to release the activation stie for SO2 derivatives through Michael addition upon exposure to acid. In vitro studies showed a remarkable selectivity of PT1 to SO₂ derivatives than other biothiols with a limit of detection as low as 62 nM. By using this acidic pH-controlled fluorescence responsiveness to SO₂, precise spatiotemporal identification of lysosomal SO2 fluctuations has been successfully performed. Furthermore, probe PT1 can be applied for monitoring SO₂ derivatives in traditional Chinese medicines.