Single Fluorescent Probe Research Articles

Pattern-based rapid and robust profiling of gut microbiota and its metabolism with a single fluorescent probe

Pattern-based rapid and robust profiling of gut microbiota and its metabolism with a single fluorescent probe

Simultaneous Genotyping of Three SNVs, rs5471, rs5472, and rs2000999 Involved in Serum Haptoglobin Levels by Fluorescent Probe-Based Melting Curve Analysis.

Haptoglobin (Hp) is a hemoglobin-binding acute-phase serum protein. Several single nucleotide variations (SNVs) within the Hp gene (HP) or Hp-related protein gene (HPR), such as rs5471 (A>C) and rs5472 (A>G) in HP promoter region and rs2000999 (G>A) in intron 2 of HRP, are suggested to correlate with the serum Hp levels. To determine these three SNVs simultaneously, a genotyping assay based on duplex dual-labeled fluorescent probes was developed. The method was then validated by analyzing genomic DNA from 121 Ghanaian and two Japanese subjects who had been previously genotyped for rs5471, rs5472, and rs2000999. Both rs5471 and rs5472 could be determined as haplotypes with a single FAM-labeled fluorescent probe, and rs2000999 could be genotyped with a HEX-labeled fluorescent probe. The results obtained with the present method were consistent with the previous results except for those of three Ghanaian subjects. All three subjects appear to have multiple HPR copy number variants characteristic of African populations, which may have led to incorrect results during previous genotyping. This method allows us to genotype these three SNVs in a relatively large number of samples, especially in African populations where rs5471 is uniquely distributed.

Fluorescence lifetime imaging: A novel approach to simultaneous visualizing lipid droplets and endoplasmic reticulum polarity in live cells and fatty liver

Cellular polarity plays an indispensable role in both physiological functions and the progression of diseases, with organelles demonstrating specific polarities that are integral to their roles. The advent of a single fluorescent probe capable of concurrently and selectively visualizing two distinct organelles through distinct fluorescence emission spectra has emerged as a powerful approach for investigating their interactions within cellular mechanisms. However, the use of changes in dual fluorescence lifetimes to monitor polarity changes in two organelles has rarely been employed, despite its potential benefits for more accurate detection of subtle changes in organelle polarity. Herein, two polarity-sensitive probes (ER-LDs-1 and ER-LDs-2) were developed. ER-LDs-2 exhibited high sensitivity to polarity alterations in both lipid droplets (LDs) and endoplasmic reticulum (ER) under various physiological and pathological conditions, including ferroptosis and endoplasmic reticulum stress, as demonstrated by distinct changes in fluorescence lifetime. ER-LDs-2 enables the monitoring of polarity changes in mouse and human normal and fatty liver tissues by fluorescence lifetime imaging. Collectively, this study presents innovative tools that hold great potential for advancing our understanding of cellular organelle polarity dynamics and the progression of associated diseases.

Dual-response fluorescent probe for lipid droplets and hypochlorous acid and imaging study in inflammation and atheromatous plaques

Simultaneously detecting two biomarkers can significantly enhance disease diagnosis accuracy and deepen our understanding of disease pathogenesis. Developing a single fluorescent probe with two independent response units for this purpose is crucial. In line with this, a novel fluorescent probe (TPA-DT-DNH) had been proposed, featuring triphenylamine and C=N groups sensitive to lipid droplets (LDs) and hypochlorous acid (HClO), respectively. Spectral results highlighted that only the co-presence of LDs and HClO could “turn-on” the fluorescence of TPA-DT-DNH, when the concentration of one substance remained constant, the fluorescence intensity linearly increased with the concentration of the other substance. The imaging results demonstrated that TPA-DT-DNH can differentiate between normal cells and cells rich in LDs when HClO was present, and can detect HClO in HeLa cells. Furthermore, dynamic changes in the inflammation process induced by LPS can be visualized in both cells and zebrafish. Additionally, significant fluorescence enhancement was observed in atheromatous plaque.

Aggregation‐based dual‐target probe for dual‐colour super‐resolution monitoring mitophagy and evaluating drugs regulating mitochondria

AbstractDeveloping single fluorescent probe for simultaneously visualizing mitophagy flux and oxidative stress with super‐resolution is highly demanded yet quite challenging. Herein, a ratiometric HClO probe AHOH is designed and synthesized which is capable of simultaneously staining lysosomes and mitochondria with red and green colour, respectively. AHOH could be selectivity oxidized by HClO, leading to a large emission blue shift (90 nm) and an over 1300‐fold enhancement of the emission ratio of Fl547nm/Fl637nm. We apply AHOH in super‐resolution microscopy and clearly visualize the dynamics of mitochondria–lysosomes interactions and the oxidative stress states upon different stimuli. Mitochondria dysfunction triggered by different drugs and genetic defect lead to elevated oxidative stress and higher levels of mitophagy. Moreover, AHOH could serve as a reliable tool for evaluating the efficacy of drugs regulating mitochondria dysfunction. This work provides a powerful dual‐colour super‐resolution imaging agent for real‐time monitoring the dynamics of organelle interactions and oxidative stress.

A portable and versatile fluorescent platform for high-throughput screening of toxic phosgene, diethyl chlorophosphate and volatile acyl chlorides

Highly toxic phosgene, diethyl chlorophosphate (DCP) and volatile acyl chlorides endanger our life and public security. To achieve facile sensing and discrimination of multiple target analytes, herein, we presented a single fluorescent probe (BDP-CHD) for high-throughput screening of phosgene, DCP and volatile acyl chlorides. The probe underwent a covalent cascade reaction with phosgene to form boron dipyrromethene (BODIPY) with bright green fluorescence. By contrast, DCP, diphosgene and acyl chlorides can covalently assembled with the probe, giving rise to strong blue fluorescence. The probe has demonstrated high-throughput detection capability, high sensitivity, fast response (within 3 s) and parts per trillion (ppt) level detection limit. Furthermore, a portable platform based on BDP-CHD was constructed, which has achieved high-throughput discrimination of 16 analytes through linear discriminant analysis (LDA). Moreover, a smartphone adaptable RGB recognition pattern was established for the quantitative detection of multi-analytes. Therefore, this portable fluorescence sensing platform can serve as a versatile tool for rapid and high-throughput detection of toxic phosgene, DCP and volatile acyl chlorides. The proposed “one for more” strategy simplifies multi-target discrimination procedures and holds great promise for various sensing applications.

Hydrogen-bond tuned conjugated architectures for nitric oxide sensing in single-benzene framework: Advances and mechanistic insights

In contrast to the conventional fluorescence enhancement resulting from the cessation of the photoinduced electron transfer effect upon capturing nitric oxide (NO) by o-phenylenediamine, we found an interesting fluorescence quench within small molecule fluorophores characterized by intramolecular hydrogen bonding. Herein, the integration of a push-pull electron system with intramolecular hydrogen bonding onto an ultra-small fluorophore was employed to fabricate a hydrogen bond-tuned single benzene core fluorescent probe with an exceptional fluorescence quantum yield of 26 %, denoted as HSC-1. By virtue of its small size and low molecular weight (mere 192 g/mol), it demonstrated superior solubility and biocompatibility. Given the optimized conditions, HSC-1 manifested extraordinary linearity in detecting NO concentrations ranging from 0.5 to 60 μM, with an outstanding detection limit of 23.8 nM. Theoretical calculations unraveled the photophysical properties of hydrogen bonding-related probe molecules and highlighted the NO sensing mechanism. This pioneering work offers an important platform for the design of small fluorescence probes only with a single benzene core applied to NO sensing, which will potentially emerge as a new frontier in the area.

A multifunctional fluorescent probe for tracking SO2 fluctuation between mitochondria and endoplasmic reticulum in living cells and zebrafish

The sulfur dioxide (SO2)-associated dynamic interplay between mitochondria (Mito) and endoplasmic reticulum (ER) is essential for intracellular homeostasis regulation. Visualizing such organelles interactions can help elucidate basic pathogenesis. However, it is hampered by challenges in developing quick response, remarkable sensitivity and high selectivity within a single fluorescent probe for simultaneous and distinct tracking of their crosstalk. Herein, we engineered a SO2-responsive Mito-ER dual-targeted fluorescent probe (MER-SO2) for dual-color visualization of Mito (red fluorescence) and ER (blue fluorescence) in living cells and in vivo. Under physiological conditions, the benzopyranium cationic structure in MER-SO2 could target Mito, following fast response to SO2 (5 s), which converted to a neutral structure, targeting to ER emitting blue fluorescence via functional fluorescence conversion. Probe MER-SO2 also displayed high sensitivity, favourable specificity (against 24 types of non-targeting analytes). Importantly, such a high-performance probe allowed in-situ and dynamic tracking of Mito-ER interactions in Mito-dysfunction living cells and zebrafish via co-localization. Furthermore, the probe enabled accurate detection of sulphite in food with recoveries rate (96–100%), and incorporation into an agarose-based portable device for SO2 assay. Taken together, this initial SO2-responsive probe represents a promising tool for discriminative tracking of functional Mito-ER interactions in living systems and holds great potential in many other fields such as food engineering.

Development of a novel fluorescent protein-based probe for efficient detection of Pb2+ in serum inspired by the metalloregulatory protein PbrR691

BackgroundThe accurate and rapid detection of blood lead concentration is of paramount importance for assessing human lead exposure levels. Fluorescent protein-based probes, known for their high detection capabilities and low toxicity, are extensively used in analytical sciences. However, there is currently a shortage of such probes designed for ultrasensitive detection of Pb2+, and no reported probes exist for the quantitative detection of Pb2+ in blood samples. This study aims to fill this critical void by developing and evaluating a novel fluorescent protein-based probe that promises accurate and rapid lead quantification in blood. ResultsA simple and small-molecule fluorescent protein-based probe was successfully constructed herein using a peptide PbrBD designed for Pb2+ recognition coupled to a single fluorescent protein, sfGFP. The probe retains a three-coordinate configuration to identify Pb2+ and has a high affinity for it with a Kd’ of 1.48 ± 0.05 × 10−17 M. It effectively transfers the conformational changes of the peptide to the chromophore upon Pb2+ binding, leading to fast fluorescence quenching and a sensitive response to Pb2+. The probe offers a broad dynamic response range of approximately 37-fold and a linear detection range from 0.25 nM to 3500 nM. More importantly, the probe can resist interference of metal ions in living organisms, enabling quantitative analysis of Pb2+ in the picomolar to millimolar range in serum samples with a recovery percentage of 96.64%–108.74 %. SignificanceThis innovative probe, the first to employ a single fluorescent protein-based probe for ultrasensitive and precise analysis of Pb2+ in animal and human serum, heralds a significant advancement in environmental monitoring and public health surveillance. Furthermore, as a genetically encoded fluorescent probe, this probe also holds potential for the in vivo localization and concentration monitoring of Pb2+.

Single Fluorescent Probe for Multiple Tasks: Illuminating Lipid Droplets and Lysosomes in Dual Channels and Distinguishing Autophagy and Apoptosis.

Lipid droplets (LDs) and lysosomes play key roles in autophagy and cell apoptosis, and the discriminative visualization of the two organelles and simultaneously of autophagy and apoptosis is very helpful to understand their internal relationships. However, fluorescent probes that can concurrently achieve these tasks are not available currently. Herein, we delicately fabricate a robust probe CAQ2 for multiple tasks: illumination of LDs and lysosomes in dual emission colors as well as discriminative visualization of cell apoptosis and autophagy. The probe exhibited both lipophilic and basic properties and displayed different emission colors in neutral and protonated forms; thus, LDs and lysosomes emitted blue and red fluorescence colors, respectively. Because of the lysosomal acidification during autophagy, CAQ2 detected autophagy with evidently enhanced red emission. Because of the lysosomal alkalization during apoptosis, CAQ2 imaged apoptosis with a drastically decreased red fluorescence intensity. With the robust probe, the autophagy under starvation and lipidless conditions was visualized, and the apoptosis induced by H2O2, ultraviolet (UV) irradiation, and rotenone treatment was successfully observed. The efficient detoxification of Na2S against rotenone treatment was successfully revealed.

A difunctional fluorescent probe to detect hypochlorite and hydrogen sulfide ions in ferroptosis and oxidative stress induced by Aspirin

HOCl and H2S frequently coexist in living cells and work in concert to maintain the redox dynamic balance in vivo. It is challenging to precisely and simultaneously trace both HOCl and H2S using a single recognition-type fluorescence probe. Here, a novel fluorescent probe (MB-HS) for the selective detection of hydrogen sulphide (H2S) and hypochlorous acid (HOCl) has been established based on the coupling of the methylene blue fraction and the indolium fraction. For HOCl, the dual-signal probe MB-HS displays quick turn-on fluorescence responses. However, for H2S, it exhibits turn-off fluorescence at 586 nm and turn-on at 513 nm, respectively. MB-HS showed many advantages such as quick response, strong selectivity, and high sensitivity. It was effectively used to detect endogenous HOCl/H2S in ferroptosis and oxidative stress induced by Aspirin in HepG-2 cells.

A single fluorescent probe to examine the dynamics of mitochondria-lysosome interplay and extracellular vesicle role in ferroptosis

Ferroptosis is a non-apoptotic form of cell death characterized by iron-dependent lipid peroxidation and glutathione (GSH) depletion. Despite recent advances, challenges remain in understanding the bidirectional interactions or interplay between organelles during ferroptosis. In this study, we aimed to understand the interplay between mitochondria (Mito) and lysosomes (Lyso) in cell homeostasis and ferroptosis. For this purpose, we designed a single fluorescent probe that marks GSH in Mito and hypochlorous acid (HOCl) in Lyso with two distinct emissions. Using this dual-targeted single fluorescent probe (9-morphorino pyronine), we detected Mito-Lyso interplay in ferroptosis. We disclosed differences in Mito-Lyso interplay depending on the induction of ferroptosis. Although erastin treatment decreased GSH, RSL3 triggered a HOCl burst, andFIN56- and FINO2-induced ferroptosis increased GSH and HOCl. Additionally, we showed that only extracellular vesicles generated during erastin-induced ferroptosis could spontaneously move and dock to neighboring cells, resulting in accelerated cell death.

In vivo deep brain multiphoton fluorescence imaging emitting at NIR-I and NIR-II and excited at NIR-IV.

Multiphoton microscopy (MPM) enables deep brain imaging. Three optical windows: NIR-I, NIR-II, and NIR-III are widely used. Recently, NIR-IV (the 2200 nm window) has been demonstrated to be the last and longest window for deep tissue MPM. However, so far MPM covers only two optical windows labeled by single fluorescent probe, one for emission and one for excitation. Here we demonstrate in vivo deep brain MPM covering three optical windows, with emission at NIR-I, NIR-II, and excitation at NIR-IV, labeled by ICG. The innovations include: (1) characterizing both 3-photon excitation and emission properties of ICG emitting at both NIR-I and NIR-II, in water, plasma, and circulating blood; (2) a home-built multiphoton microscope with simultaneous dual channel detection, with which we demonstrate deep brain MPM 950 μm (NIR-I) and 850 μm (NIR-II) into the mouse brain in vivo, verifying that multi-optical window MPM is promising for deep brain imaging.

Dual-Labeled Single Fluorescent Probes for the Simultaneous Two-Color Visualization of Dual Organelles and for Monitoring Cell Autophagy.

Dual-labeled single fluorescent probes are powerful tools for studying autophagy on the molecular scale, yet their development has been hampered by design complexity and a lack of valid strategies. Herein, for the first time, we introduce a combinatorial regulation strategy to fabricate dual-labeled probes for studying autophagy by integrating the specific organelle-targeting group and the functional fluorescence switch into a pentacyclic pyrylium scaffold (latent dual-target scaffold). For proof of concept, we prepared a range of dual-labeled probes (TMOs) that display different emission colors in duple organelles. In these probes, TMO1 and TMO2 enabled the simultaneous two-color visualization of the lysosomes and mitochondria. The other probes (TMO3 and TMO4) discriminatively targeted lysosomes/nucleolus and lysosomes/lipid droplets (LDs) with dual-color emission characteristics, respectively. Intriguingly, by simply connecting the endoplasmic reticulum (ER) targeting group to the pentacyclic pyrylium scaffold, we created the first dual-labeled probe TMO5 for simultaneously labeling lysosomes/ER in distinctive fluorescent colors. Subsequently, using the dual-labeled probe TMO2, drug-induced mitophagy was successfully recorded by evaluating the alterations of multiple mitophagy-related parameters, and the mitophagy defects in a cellular model of Parkinson's disease (PD) were also revealed by simultaneous dual-color/dual-organelle imaging. Further, the probe TMO4 can track the movement of lysosomes and LDs in real time and monitor the dynamic process of lipophagy. Therefore, this work not only presents attractive dual-labeled probes to promote the study of organelle interactions during autophagy but also provides a promising combinatorial regulation strategy that may be generalized for designing other dual-labeled probes with multiple organelle combinations.

Simultaneous two-color visualization of lipid droplets and lysosomes for cell homeostasis monitoring using a single fluorescent probe.

Lipid droplets (LDs) and lysosomes are vital organelles that play crucial roles in various physiological and pathological processes. However, simultaneous two-color visualization of these two organelles using a single probe for cell homeostasis monitoring remains a challenge due to the lack of rational design strategies. To address this issue, we have developed an aggregation-induced emission (AIE) fluorescent probe named TPE-NDI-Mor with an electron donor (D)-acceptor (A) structure, which can stain both LDs and lysosomes with high selectivity through green and red fluorescence imaging, respectively. A detailed mechanism study revealed that TPE-NDI-Mor, with a twisted intramolecular charge transfer (TICT) effect, shows a high affinity for a polar microenvironment. Additionally, the probe also demonstrates good stability, high anti-interference performance and a large Stokes shift, making it suitable for visualizing cell homeostasis and further disease diagnosis by tracking the dynamic changes of LDs and lysosomes.

A single mitochondria-targetable fluorescent probe for visualizing cysteine and glutathione in ferroptosis of myocardial ischemia/reperfusion injury

Ferroptosis plays an important role in the early stage of myocardial ischemia/reperfusion (MI/R) injury, which is closely associated with the antioxidant damage of mitochondrial cysteine (Cys)/glutathione (GSH)/glutathione peroxidase 4 (GPX4) axis. Visualization of Cys and GSH in mitochondria is meaningful to value ferroptosis and further contributes to understanding and preventing MI/R injury. Herein a mitochondria-targetable thiols fluorescent probe (MTTP) was designed and synthesized based on sulfonyl benzoxadiazole (SBD) chromophore with a triphenylphosphine unit as the mitochondria-targeted functional group. Cys and GSH can be differentiated by MTTP with two distinguishable emission bands (583 nm and 520 nm) through the controllable aromatic substitution-rearrangement reaction. Importantly, MTTP is capable of monitoring ferroptosis and its inhibition by measuring mitochondrial Cys and GSH. MTTP was also employed to non-invasively detect ferroptosis during oxygen and glucose deprivation/reoxygenation (OGD/R)-induced MI/R injury in H9C2 cells. In a word, MTTP provides a visual tool that can simultaneously detect Cys and GSH to monitor ferroptosis processes during MI/R injury, which helps for more deeper understanding of the role of ferroptosis in MI/R injury-related diseases.

A fluorescent probe based on carbon quantum dots with spectral selectivity for sensitive detection of Cr(VI) and Hg(II) in environmental waters

In this paper, fluorescent carbon dots were prepared by hydrothermal method using 3,5-dihydroxybenzoic acid and l-Arginine as precursors. The synthesized CDs have excitation-dependent emission with emission peaks at 354 nm and 460 nm, corresponding to the optimal excitation wavelengths of 295 nm and 330 nm, respectively. CDs have good dispersion and resistance to photobleaching in aqueous solution, and can maintain stable fluorescence in highly concentrated salt environment. Based on the inner filter effect, Cr(VI) with strong absorption can shield the excitation light of CDs at 295 nm and absorb the emission fluorescence at 354 nm simultaneously. Due to the intermolecular electrostatic interaction, Hg(II) can coordinate with CDs surface groups and cause static quenching, which significantly reduces the fluorescence intensity of CDs emitted at 460 nm. The linear ranges were 0.1–2 μM and 0.4–5 μM for Cr(VI) and Hg(II) detection, respectively, and limits of detection were 0.024 μM and 0.084 μM. Additionally, the probe was successfully applied to tap water and lake water samples. With high sensitivity and spectral selectivity, this sensing strategy realized the detection of two heavy metal ions by a single fluorescent probe, demonstrating its great potential in the field of environmental metal analysis.

A multifunctional near-infrared fluorescence probe with multi-reactive sites for real-time tracking Cys to SO2 and GSH to SO2 metabolism in vivo

Reactive sulfur species (RSS), including sulfur dioxide (SO2), glutathione (GSH), homocysteine (Hcy) and cysteine (Cys), and their associated metabolic processes, are essential for biological systems to maintain normal life activities. However, due to the similarity of their chemical properties and the diversity of the metabolic pathways, using a single fluorescence probe to differentiate RSS and monitor the Cys-SO2 and GSH-SO2 metabolic pathways in real time is rather difficult. Herein, we developed a multifunctional near-infrared (NIR) fluorescence probe NIR-COU with multiple response sites, in which aldehyde and ether groups served as the specific recognition sites for GSH and Cys/Hcy, and active CC as the specific response site for SO2. Moreover, CN+ with another new reactive site formed after the reaction of GSH with the probe, together with CC was applied as the specific reactive site for SO2 for real-time monitoring of Cys to SO2 and GSH to SO2 metabolic processes. The structures of the NIR-COU and intermediates were characterized by NMR and HRMS. Based on the FRET mechanism, the NIR-COU exhibited different fluorescence signals under different excitations (385, 460 and 580 nm), not only for GSH (enhanced green and NIR emissions), Cys/Hcy, (enhanced blue and NIR emissions) and SO2 (enhanced green emissions only), but also ratiometric fluorescence responses to the metabolic pathways of GSH to SO2 and Cys to SO2. Moreover, NIR-COU could also be utilized for imaging GSH, Cys/Hcy, SO2 and visual tracking metabolic pathways from Cys to SO2 and GSH to SO2 in living cells as well as zebrafish.

Biocompatible, Europium-Doped Fluorapatite Nanoparticles as a Wide-Range pH Sensor.

The development of a simple, biocompatible, pH sensor with a wide range of detection, using a single fluorescent probe is highly important in the medical field for the early detection of diseases related to the pH change of tissues and body fluids. For this purpose, europium-doped fluorapatite (FAP: Eu) nanoparticles were synthesized using the coprecipitation method. Doping with the rare earth element europium (Eu) makes the non-luminescent phosphate mineral fluorapatite, luminescent. The luminous response of the sample upon dissolution in hydrochloric acid (HCl), in highly acidic to weakly basic media, makes it a potential pH sensor. A linear variation was observed with an increase in pH, in both the total intensity of emission and the R-value or the asymmetry ratio. The ratiometric pH sensing enabled by the variation in R-value makes the sensor independent of external factors. The structural, optical, and photoluminescent (PL) lifetime analysis suggests a particle size-dependent pH sensing mechanism with the changes in the coordinated water molecules around the Eu3+ ion in the nanoparticle. Given its exceptional biocompatibility and pH-dependent fluorescence intensity for a wide range of pH from 0.83 to 8.97, the probe can be used as a potential candidate for pH sensing of biological fluid.

A Single Organic Fluorescent Probe for the Discrimination of Dual Spontaneous ROS in Living Organisms: Theoretical Approach.

Single-organic-molecule fluorescent probes with double-lock or even multi-lock response modes have attracted the attention of a wide range of researchers. The number of corresponding reports has rapidly increased in recent years. The effective application of the multi-lock response mode single-molecule fluorescent probe has improved the comprehensive understanding of the related targets' functions or influences in pathologic processes. Building a highly efficient functional single-molecule fluorescent probe would benefit the diagnosis and treatment of corresponding diseases. Here, we conducted a theoretical analysis of the synthesizing and sensing mechanism of this kind of functional single-molecule fluorescent probe, thereby guiding the design and building of new efficient probes. In this work, we discuss in detail the electronic structure, electron excitation, and fluorescent character of a recently developed single-molecule fluorescent probe, which could achieve the discrimination and profiling of spontaneous reactive oxygen species (ROS, •OH, and HClO) simultaneously. The theoretical results provide insights that will help develop new tools for fluorescent diagnosis in biological and medical fields.