Dual-emission Probe Research Articles

Biotin-Modified PEGylated Protein-Stabilized gold Nanoclusters: A strategy for selective and efficient intracellular transport of glucose oxidase for glucose detection

Although bovine serum albumin-stabilized gold nanoclusters (BSA-AuNCs) have various applications in biological environments., limited research has been carried out on their potential for intracellular delivery of enzymes and highly selective intracellular detection of specific analytes. We developed biotin-capped polyethylene glycol (PEG)-functionalized bovine serum albumin-stabilized gold nanoclusters (biotin-AuNCs) for enhanced optical properties and bioimaging applications. Biotin-AuNCs exhibited superior performance to BSA-AuNCs, with higher quantum yield (∼14 %), improved photostability, and enhanced resistance to trypsin digestion. These nanoclusters enabled selective imaging of biotin receptor-positive cells such as HeLa and MCF-7, while forming nanocomposites (GOx-AuNCs) with avidin-glucose oxidase (GOx) for intracellular glucose detection. The GOx-AuNCs showed selective imaging capabilities and facilitated glucose sensing through GOx-catalyzed H2O2 production, leading to fluorescence quenching of the biotin-AuNCs. This method provided a reliable indicator for intracellular glucose levels, establishing a linear calibration curve between GOx-AuNC-labeled cells’ luminescence intensity and intracellular glucose concentration. Additionally, we developed dual-emission probes by modifying red-emitting GOx-AuNCs with green-emitting BDP-FL for ratiometric imaging of intracellular glucose concentration, enhancing glucose sensing precision in biological environments.

Dual-emission Tb-based coordination polymer as a ratiometric fluorescence probe for the detection of phosphate.

A simple, sensitive dual-emission probe was developed for the detection of phosphate (Pi). The probe Tb-BTB/DPA was synthesized by mixing dual-ligand, 1,3,5-tri(4-carboxyphenyl) benzene (H3BTB) and dipicolinic acid (DPA), with metal ions Tb3+ in ethanol-water solution at 40℃ for 2h. Tb-BTB/DPA exhibits two emission peaks, the emission at 362nm is attributed to H3BTB, an energy transfer between Tb3+ nodes, and DPA further enhances the fluorescence of Tb3+ at 544nm. Pi competes with ligand H3BTB to coordinate Tb3+, resulting in partial collapse of the Tb-BTB/DPA structure and interrupting the electron transfer between H3BTB and Tb3+. Therefore, the emission at 362nm is enhanced, while the emission at 544nm is unchanged, and a ratiometric fluorescence method is developed to detect Pi. Tb-BTB/DPA exhibits good linearity within the Pi concentration range (0.1-50µmol/L), and the detection limit was 25.8nmol/L. This study provides a new way to prepare probes with dual emission sensing properties.

Viscosity-modulated intramolecular excitation energy transfer for mitochondria-targeted sensing and photokilling

Viscosity is a key parameter of mitochondria that related to several diseases. To design a probe acting as both the mitochondria-targeted viscosity sensor and photosensitizer is meaningful for diagnosis and therapies. Herein, we report a ratiometric viscosity sensor with dual-emission mediated by excitation energy transfer (EET) effect, which can detect the mitochondrial viscosity and serve as an efficient photosensitizer to eradicate cancer cells. The probe comprises of a blue-emitting imidazole moiety as the energy donor and a green-emitting boron dipyrromethene (BODIPY) core as the energy acceptor, respectively. Via viscosity-modulated EET effect, the dual-emissive probe can sensitively detect the medium viscosity in a ratiometric model. Directed by imidazole as the targeting factor, the probe can specifically accumulate in mitochondria and ratiometrically determine the intracellular viscosity increase induced by Nystatin treatment. Furthermore, it also exhibits light-triggered reactive oxygen species (ROS) generation including singlet oxygen and superoxide radical, which can efficiently kill the cancer cells under white light illumination. Thus, this work provides a versatile and promising candidate for disease diagnosis and phototherapy.

Anti-cancer drug axitinib: a unique tautomerism-induced dual-emissive probe for protein analysis.

A commercial anti-cancer drug, axitinib, exhibits very stable dual emissions for discrimination of human serum albumin.

Construction of a Dual-Emissive Probe for Discriminative Visualization of Lysosomal and Mitochondrial Dysfunction.

Discriminatively visualizing mitochondrial and lysosomal dysfunction is crucial for an in-depth understanding of cell apoptosis regulation and relative biology. However, fluorescent probes for the separate visualization of lysosomal and mitochondria damages have not been reported yet. Herein, we have constructed a fluorescent probe [2-(4-hydroxystyryl)-1,3,3-trimethyl-3H-indol-1-ium iodide (HBSI)] for labeling mitochondria and lysosomes in dual emission colors and discriminatively imaging mitochondrial and lysosomal damage in two different sets of fluorescent signals. In living cells, HBSI targeted both lysosomes and mitochondria to give green and red emission, respectively. During mitochondrial damages, HBSI immigrated into lysosomes, and the red emission decreased. During lysosomal damage, HBSI immigrated into mitochondria, and the green emission decreased. With the robust probe, the different damaging sequences of mitochondria and lysosomes under different amounts of H2O2 and chloral hydrate have been revealed. The sequential damage of lysosomes and mitochondria during cell apoptosis induced by rotenone, paclitaxel, and colchicine has been discovered. Furthermore, the regulation of mitochondria, lysosome, and their interplay during autophagy was also observed with the probe.

Fluorescent/Phosphorescent Dual-Emissive Probe for Simultaneous and Independent Sensing of Intracellular Hypochlorite and pH via Time-Resolved Luminescence Imaging.

In this work, we present a dual-emissive polymeric probe capable of simultaneous and independent sensing of intracellular hypochlorite and pH. The probe integrates an anthracene-carboxamide-based fluorescent compound with a phosphorescent iridium(III) complex. The fluorescence and phosphorescence spectra are significantly overlapped at 500 to 650 nm, but their lifetimes differ substantially at 14.8 and 1113.6 ns, respectively, thereby allowing for monitoring in different time intervals through time-resolved luminescence analysis. The fluorescence is quenched upon oxidation by hypochlorite, whereas an acidic environment favors protonation of pyridyl groups on the iridium(III) complex, which causes phosphorescence quenching. During hypochlorite sensing, a linear plot of the fluorescence/phosphorescence ratio over hypochlorite concentration was obtained. During pH sensing, the pK a value was calculated to be about 4.6. The utilization of this probe for imaging cellular endogenous hypochlorite and intracellular pH values was demonstrated. The short-lived and long-lived signals were successfully extracted via time-resolved luminescence imaging microscopy, thereby allowing independent, simultaneous, and quantitative analysis of fluorescence and phosphorescence signals and their response toward hypochlorite and pH values. This study offers potential implications for the development of biosensing and bioimaging applications and paves the way for the simultaneous multiplexed detection of physiological and pathological activities within living cells.

A core–shell structure ratiometric fluorescent probe based on carbon dots and Tb3+ for the detection of anthrax biomarker

A novel core–shell structure ratiometric fluorescent probe was developed, which can selectively and sensitively detect 2,6-dipicolinic acid (DPA) as an anthrax biomarker. Carbon dots (CDs) was embedded into SiO2 nanoparticles, which was acted as an internal reference signal. Tb3+ with green emission was connected to the carboxyl functionalized SiO2, which was acted as a responsive signal. With the addition of DPA, the emission of CDs at 340 nm was unchanged, while the fluorescence of Tb3+ at 544 nm was enhanced by the antenna effect. In the concentration range of 0.1–2 μM, the fluorescence intensity ratio of I544/I340 showed a good linear relationship with the concentration of DPA, and the limit of detection (LOD) was 10.2 nM. In addition, the dual-emission probe showed an obvious fluorescence color change from colourless to green with increasing DPA under UV light, which enabled visual detection.

A dual-signal fluorometric and colorimetric sensing platform based on gold-platinum bimetallic nanoclusters for the determination of β-galactosidase activity

Herein, a novel dual-signal sensing system for the determination of β-galactosidase (β-Gal) activity was established, which was based on a dual-emission probe assembled from gold-platinum bimetallic nanoclusters (Au–Pt NCs) and rhodamine B. Under the catalysis of β-Gal, 4-nitrophenyl β-D-galactopyranoside (PNPG) was rapidly hydrolyzed to generate p-nitrophenol (PNP), which has an obvious UV absorption peak at 400 nm. The hydrolyzed product PNP can quench the fluorescence of Au–Pt NCs effectively by inner filter effect (IFE), and PNP had no impact on the fluorescence of rhodamine B, which will change the emission intensity ratio of Au–Pt NCs and rhodamine B. Therefore, the ratiometric fluorescent and colorimetric dual-signal sensor based on Au–Pt NCs and rhodamine B was successfully constructed for sensitive detection of β-Gal activity. The linear detection range for the ratiometric fluorescence and colorimetric methods were 2.5–25 U/L and 15–55 U/L with detection limits of 1.2 U/L and 5.2 U/L, respectively. The developed assay method has been used for quantitative detection of β-Gal in spiked serum samples and showed good performance. And the detection platform has high reliability and excellent selectivity, which opens a new avenue for the further application of Au–Pt NCs in chemical sensing and biological analysis.

Kill two birds with one stone: Ratiometric sensing of phosphate via a single-component probe with fluorescence-scattering dual-signal response behavior

Ratiometric fluorescence sensors gain stronger anti-interference ability via self-calibration. Nevertheless, ratiometric analysis of phosphate (Pi) still faces problems such as complicated construction process of dual emission probes and possible interferences from outputting mono-category fluorescent signal. Herein, we propose a “kill two birds with one stone” strategy to address these challenges, by simply introducing a single-component probe, porphyrin paddlewheel framework-3 (PPF-3) nanosheets without modification, encapsulation or complex, to integrate fluorescence (FL)-second-order scattering (SOS) dual-signal for ratiometric detection of Pi. PPF-3 nanosheets are constructed by coordination of Co2+ with 5,10,15,20-tetrakis(4-carboxyl-phenyl)-porphyrin (TCPP) ligands, displaying weak FL and strong SOS, two different and independent signals. In the response system to Pi, Co2+ and TCPP serve as the recognition element and signal unit, respectively. After interacting with Pi, the high affinity for Co2+ makes Pi snatch Co2+ from the PPF-3 nanosheets, causing their structure disassembly (SOS decrease) and TCPP release (FL increase). Finally, the FL-SOS ratiometric platform is successfully employed to access Pi in real water samples. Synchronous collection of FL and SOS from the single-component probe provides a simpler and more efficient way on ratiometric sensor design as well as a new useful technique for monitoring target-induced aggregation and disaggregation behavior.

Intramolecular hydrogen bond-tuned thermal-responsive carbon dots and their application to abnormal body temperature imaging

A steric hindrance strategy was used to prepare intramolecular hydrogen bond–controlled thermosensitive fluorescent carbon dots (CDs) via the solvothermal treatment of o-phenylenediamine respectively with three dihydroxybenzene isomers. The CDs obtained from different isomers have very similar morphology, surfaces, and photophysical properties but exhibited different thermal sensitivities. Meanwhile, the orange-emitting CDs (p-CDs) obtained from o-phenylenediamine and p-hydroquinone exhibited an optimal thermal sensitivity of 1.1%/°C. Comprehensive experimental characterizations and theoretical calculations revealed that even a small difference in substituent locations in the phenyl ring of the precursors can considerably affect the formation of intramolecular hydrogen bonds and that the CDs with strong intramolecular hydrogen bonds exhibited poor thermosensitivity. The p-CDs were incorporated with reference CDs (B-CDs) that exhibited heating–quenching blue emission through electrostatic self-assembly to construct a dual-emission probe (p-CDs/B-CDs), which exhibited a thermal sensitivity of 2.0%/°C. Test strips based on the p-CDs/B-CDs were prepared to measure temperature fluctuations based on sensitive and instant fluorescence color evolution. Further, this fluorescent colorimetry was successfully applied to a test strip–integrated wearable wristband to measure the body temperature. This study establishes an inherent relationship between precursors and the resulting intramolecular hydrogen bonds for precisely tuning the thermal sensitivity of CDs. It also offers a visual quantitative strategy for the early warning of abnormal body temperatures.

Simultaneous detection of acute myocardial infarction -related miR-199a and miR-499 based on a dual-emission CdTe fluorescent probe and T7 exonuclease-assisted signal amplification

The early diagnosis of acute myocardial infarction (AMI) was a tough nut in both clinical medicine and forensic science since the occurrence of gross changes needed at least 12 h. Compared to other diagnostic methods, microRNA was reported as an earlier cardiac biomarker with higher sensitive. In this work, we designed a novel fluorescent analytical strategy using a dual-emission probe based on T7 Exo-assisted target recycling amplification for simultaneous detection of AMI-related miR-199a and miR-499, which could indicate myocardial ischemia or damage in AMI. The dual-emission probe was conducted by yellow-emission and red-emission CdTe quantum dots capped with beta-cyclodextrin. The florescence of probe was firstly quenched by graphene oxide (GO) and then recovered in the presence of target miR-199a and miR-499. Under the optimized conditions, the limit of detection as low as 0.1 pM was obtained and the overall test could be performed within 50 min. Besides, the simultaneous detections of microRNAs in spiked serum samples were achieved with the recovery of 81.3–109.6%, which suggested the potential method for diagnosis in practical. The advantages of this method include easy operation, high accuracy and low labor cost, which makes point-of-care test possible both in hospital and forensic application.

A highly water-stable dual-emission fluorescent probe based on Eu3+-loaded MOF for the simultaneous detection and quantification of Fe3+ and Al3+ in swine wastewater

A novel dual-emissive Eu3+-loaded metal–organic framework (MOF) is designed and successfully fabricated by introducing 2-hydroxyterephthalic acid (H2BDC-OH) and Eu3+ ions into an UiO-66-type MOF material. The obtained MOF, here referred to as EuUCH, exhibits dual emission at 450 and 614 nm, and both emissions are quite stable in aqueous media in the pH range of 4–11. EuUCH is characterized by a high selectivity and sensitivity toward Fe3+ and Al3+, which yield different responsive modes. The two emissions of EuUCH are quenched by Fe3+; by contrast, only the emission at 450 nm is quenched by Al3+ showing a ratiometric fluorescence signal. More importantly, as there is no clear interference between the signals of Fe3+ and Al3+, EuUCH is successfully utilized for the simultaneous detection of Fe3+ and Al3+ in their mixtures. In addition, the simultaneous quantification of Fe3+ and Al3+ is achieved in more complicated swine wastewater with good recoveries. This work provides a water-stable dual-emissive probe and the possibility to achieve the simultaneous quantification of Fe3+ and Al3+ in complicated environment wastewater.

A smartphone-integrated light-up lanthanide fluorescent probe for the visual and ratiometric detection of total phosphorus in human urine and environmental water samples

Phosphate (Pi) plays an essential role in aquatic ecosystems as well as in physiological processes. Here, a dual-emission probe for the sensitive, specific and visual analysis of Pi is fabricated by coordinating Eu3+ with luminol and 2,6-pyridinedicarboxylic acid (DPA). Pi can significantly enhance the characteristic fluorescence of Eu3+ at 615 nm by promoting energy transfer from DPA to Eu3+ and reducing the quenching effect of water molecule, luminol with inherent emission at 423 nm further enhances the Eu3+ fluorescence. Accordingly, ratiometric detection of Pi can be achieved with the fluorescence ratio F615/F423 as a function of Pi concentration. Linearity between F615/F423 and Pi concentration in the range of 0.1–25 μM is shown, and the limit of detection (LOD, 3σ/K) for Pi is 0.027 µM. In addition, a continuous change in the fluorescence color of the probe from blue to red is observed with increasing Pi concentration under a UV lamp, and a smartphone-based visual method is used for the convenient and effective semi-quantitative determination of Pi. The dual-emission probe has been successfully applied to ratiometric and visual analysis of Pi in human urine and environmental water samples, and adequate results are obtained.

Lanthanide metal doped organic gel as ratiometric fluorescence probe for selective monitoring of ciprofloxacin

In this experiment, a novel ratio fluorescent gel probe based on double rare earth metal doped ‘Terbium-Europium-metal organic gel’ Tb-Eu-MOG was designed and successfully achieved the analysis and monitoring of ciprofloxacin (CIP). The dual emission probe (Tb-Eu-MOG) sensing is based on adjusting the energy and electronic conversion between the boronic acid-based ligand and Tb3+, Eu3+ ions. The 3,5-dicarboxybenzeneboronic acid (DBA) organic ligand not only used as the “antenna” to magnify the luminescence of Tb3+ and Eu3+ but also given strong support for the construction of the metal organic gel three-dimensional system. Dual fluorescence emission from lanthanide elements were generated at 545 nm and 615 nm under the excitation of 295 nm, respectively. Based on the photo-induced charge transfer (PET) and fluorescence resonance energy transfer (FRET) between the electron-donating group and the electron-withdrawing functional group, it provided a good test environment for the specific and high-sensitivity detection of CIP. Attributable to the porous structure formed by the intersecting of metal organic gel fiber bundles, as well as mutual attraction between the boron atomic orbital and CIP, the novel polymer probe exhibited outstanding characteristics for analysis of CIP including, fluorescence stabilization, rapid response, high selectivity and sensitivity. The doping ratiometric fluorescence monitoring strategy provided an alternative sensing method for the rapid and sensitive analytical determination of ciprofloxacin in complex biological matrix, and the reasonable doping of MOG provided some enlightenment.

A dual-emission fluorescence-enhanced probe for hydrogen sulfide and its application in biological imaging

A fluorescence-enhanced probe with unique dual-channel emissions was designed for the detection and bioimaging of hydrogen sulfide.

Synthesis of gold nanoclusters-loaded lysozyme nanoparticles for ratiometric fluorescent detection of cyanide in tap water, cyanogenic glycoside-containing plants, and soils

The modification of protein-stabilized gold nanoclusters with fluorophores has been intensively applied for the ratiometric detection of biomolecules, metal ions, and anions. This study developed a straightforward strategy to prepare lysozyme nanoparticle-encapsulated gold nanoclusters (LysNP-AuNCs) as a dual-emission probe for the ratiometric sensing of cyanide through fluorescence resonance energy transfer (FRET) without the conjugation of additional fluorophores. The reduction of gold ion precursors with lysozyme generated lysozyme-stabilized AuNCs under an alkaline pH, which were demonstrated to self-assemble into nanoaggregates during the formation of AuNCs. The aggregated lysozyme molecules on the AuNCs were treated with glutaraldehyde, triggering the conversion of the aggregated lysozymes into blue-emitting lysozyme nanoparticles. As a result, the AuNCs were well distributed inside a single lysozyme nanoparticle, as demonstrated by transmission electron microscopy. The presence of cyanide triggered the etching of the AuNCs in the LysNP-AuNCs, leading to the suppression of FRET from lysozyme nanoparticle to AuNCs. The LysNP-AuNC probe was implemented for FRET detection of cyanide with a linear range of 3–100 μM. Additionally, the selectivity of the LysNP-AuNC probe for cyanide toward other anions was remarkably high. The practicality of the proposed probe was evaluated by quantifying cyanide in tap water and soils and monitoring the liberation of hydrogen cyanide from cyanogenic glycoside-containing foods.

Dual-Emission Fluorescence Probe Based on CdTe Quantum Dots and Rhodamine B for Visual Detection of Mercury and Its Logic Gate Behavior.

It is urgent that a convenient and sensitive technique of detecting Hg2+ be developed because of its toxicity. Conventional fluorescence analysis works with a single fluorescence probe, and it often suffers from signal fluctuations which are influenced by external factors. In this research, a novel dual-emission probe assembled through utilizing CdTe quantum dots (QDs) and rhodamine B was designed to detect Hg2+ visually. Only the emission of CdTe QDs was quenched after adding Hg2+ in the dual-emission probe, which caused an intensity ratio change of the two different emission wavelengths and hence facilitated the visual detection of Hg2+. Compared to single emission QDs-based probe, a better linear relationship was shown between the variation of fluorescence intensity and the concentration of Hg2+, and the limit of detection (LOD) was found to be11.4 nM in the range of 0–2.6 μM. Interestingly, the intensity of the probe containing Hg2+ could be recovered in presence of glutathione (GSH) due to the stronger binding affinity of Hg2+ towards GSH than that towards CdTe QDs. Based on this phenomenon, an IMPLICATION logic gate using Hg2+/GSH as inputs and the fluorescence signal of QDs as an output was constructed.

Aggregation-enhanced emission of metal nanoclusters triggered by peptide self-assembly and application in chymotrypsin inhibitor screening

Herein, a new strategy is developed to induce the aggregation of glutathione-capped gold nanoclusters (GSH-AuNCs) by self-assembly of peptide. The diphenylalanine peptide (L-Phe-l-Phe, FF) self-assembles in aqueous solution into the nano-net structure (FF-Nanonet, FFN). With self-assembly of FF, GSH-AuNCs aggregate in the network of FFN to form a dual-emission probe, called FF-N@GSH-AuNCs. One emission is from the self-assembled FFN at 470 nm, and the other one is the aggregation-induced enhanced emission (AIEE) of the GSH-AuNCs at 575 nm. Due to the hydrolysis of FF by chymotrypsin (CHT), the peptide bond on the carboxyl side of the phenylalanine is cleaved and the AIEE from GSH-AuNCs becomes faint because the released nanoclusters are dispersed in the solution again. Therefore, FF-N@GSH-AuNCs can be applied to screen and evaluate the CHT inhibitors. The inhibitory effects of four ingredients from natural products (i.e. epigallocatechin gallate (EGCG), tannic acid, oleanolic acid, and ursolic acid) on CHT activity have been studied. Oleanolic acid and ursolic acid are invalid. The half maximal inhibitory concentration (IC 50) values of EGCG and tannic acid are 13.9 and 19.2 μM, respectively. The screening results show that these two compounds have significant inhibitory effects on CHT.

NAD(P)H Quinone Oxidoreductase-1 in Organ and Tumor Tissues: Distinct Activity Levels Observed with a Benzo-rosol-Based Dual-Excitation and Dual-Emission Probe.

NAD(P)H quinone oxidoreductase-1 (NQO1), a protective enzyme against cellular oxidative stress, is expressed abnormally high in solid tumors and thus recognized as a cancer biomarker. To develop a fluorescent NQO1 probe with practicality, we investigated benzo-rosol fluorophores linked with a known self-immolative quinone substrate. Four probe candidates exhibited ratiometric sensing behavior toward the enzyme, satisfying our orbital mismatch stratagem proposed before, under dual-excitation and dual-emission conditions that alleviate the spectral overlap issue commonly observed with the ratiometric probes based on intramolecular charge-transfer change. Among the candidates, two ester-linked compounds exhibited hydrolytic instability to water or an esterase, discouraging us to develop such ester-linked probes. One ether-linked, hydrolytically stable probe provided brighter cellular fluorescence than the other and thus was applied to ratiometric imaging of NQO1 in cells and tissues. We found that the enzyme activity levels are much different in organ tissues: stomach (56), kidney (22), colon (9.8), testis (7.8), bladder (5.6), lung (1.2), and muscle (1.0). Furthermore, a markedly high enzyme level (14.6-fold) was observed in a xenograft tumor tissue compared with that in a normal tissue, which suggests that such an NQO1 probe is promising for cancer diagnosis and for studying the enzyme-associated biology.

A POSS-assisted fluorescent probe for the rapid detection of HClO in mitochondria with a large emission wavelength in dual channels.

Hypochlorous acid (HClO) is closely related to many diseases and is an inevitable part of the physiological processes. It is significant to detect HClO in mitochondria for getting meaningful physiological and pathological information. However, adequate tools to detect HClO with emissions in two channels are rarely reported. To achieve this target, in this work, a "turn-off" visual and near infrared (NIR) fluorescent dual emission probe D6 based on polyhedral oligomeric silsesquioxanes (POSS) was successfully designed and synthesized. D6 showed high selectivity and sensitivity to HClO. Notably, the emission wavelength of D6 reached 820 nm due to the assistance of the POSS cage. In addition, bioimaging experiments clearly showed that probe D6 promoted the visualization of exogenous and endogenous HClO in living HepG2 cells and zebrafish models.