Fluorescent Probe Research Articles

Promotion and Detection of Cell-Cell Interactions through a Bioorthogonal Approach.

Manipulation of cell-cell interactions via cell surface modification is crucial in tissue engineering and cell-based therapy. To be able to monitor intercellular interactions, it can also provide useful information for understanding how the cells interact and communicate. We report herein a facile bioorthogonal strategy to promote and monitor cell-cell interactions. It involves the use of a maleimide-appended tetrazine-caged boron dipyrromethene (BODIPY)-based fluorescent probe and a maleimide-substituted bicyclo[6.1.0]non-4-yne (BCN) to modify the membrane of macrophage (RAW 264.7) and cancer (HT29, HeLa, and A431) cells, respectively, via maleimide-thiol conjugation. After modification, the two kinds of cells interact strongly through inverse electron-demand Diels-Alder reaction of the surface tetrazine and BCN moieties. The coupling also disrupts the tetrazine quenching unit, restoring the fluorescence emission of the BODIPY core on the cell-cell interface, and promotes phagocytosis. Hence, this approach can promote and facilitate the detection of intercellular interactions, rendering it potentially useful for macrophage-based immunotherapy.

Delineating a tailor-made fluorescent probe designed for the selective detection of tyrosinase.

A dicyanoisophorone based fluorescent probe (E)-2-(3-(4-hydroxystyryl)-5,5-dimethylcyclohex-2-en-1-ylidene)malononitrile (DCIP-OH) was developed for the selective sensing of tyrosinase in apple extract and live cells. The probe was obtained by the condensation of 2-(3,5,5-trimethylcyclohex-2-en-1-ylidene)malononitrile with 4-hydroxybenzaldehyde. Upon interaction with tyrosinase, the probe exhibited absorbance switching from 417 nm to 357 nm, accompanied by a slight increase in absorption value and an isosbestic point observed at 373 nm. Additionally, a reduction in emission intensity at 592 nm was observed. Furthermore, we successfully employed the probe for sensing of tyrosinase in apple extract and conducted inhibition studies by using kojic acid. LOD was determined to be ~0.4 nM. Moreover, the biocompatible nature of DCIP-OH enabled its effective localization in epithelial-like melanoma cells, B16F10, where it demonstrated successful fluorescent probing of intracellular tyrosinase.

Highly-Selective fluorescent Fe3O4@PPy aptasensor.

Label-free nucleic acid fluorescent probes are gaining popularity due to their low cost and ease of application. However, the primary challenges associated with label-free fluorescent probes stem from their tendency to interact with other biomolecules, such as RNA, proteins, and enzymes, which results in low specificity. In this work, we have developed a simple detection platform that utilizes Fe3O4@PPy in combination with a label-free nucleic acid probe, 1,1,2,2-tetrakis[4-(2-bromo-ethoxy)phenyl]ethene (TTAPE) or Malachite Green (MG), for highly selective detection of metal ions, acetamiprid, and thrombin. Fe3O4@PPy not only adsorbs aptamers through electrostatic interactions, π-π bonding, and hydrogen bonding, but also quenches the fluorescence of the TTAPE/MG. Upon the addition of target compounds, the aptasensor separates from Fe3O4@PPy through magnetic separation. Moreover, by changing different aptamers, the aptasensor was applied to detect metal ions, acetamiprid, and thrombin, with the turned-on photoluminescence (PL) emission intensity recorded and showing linearity to the concentrations of targets. The robustness of method was demonstrated by applying it to real samples, which included vegetables (for detecting acetamiprid with LODs of 0.02 and 0.04ng/L), serum samples (for detecting thrombin with LODs of 5.5 and 4.3nM), and water samples (for detecting Pb2+ with an LOD of 0.17nM). Therefore, due to its impressive selectivity and sensitivity, the Fe3O4@PPy aptasensor could be utilized as a universal detection platform for various clinical and environmental applications.

Flow-based bioconjugation of coumarin phosphatidylethanolamine probes: Optimised synthesis and membrane molecular dynamics studies.

A series of phosphatidylethanolamine fluorescent probes head-labelled with 3-carboxycoumarin was prepared by an improved bioconjugation approach through continuous flow synthesis. The established procedure, supported by a design of experiment (DoE) set-up, resulted in a significant reduction in the reaction time compared to the conventional batch method, in addition to a minor yield increase. The characterization of these probes was enhanced by an in-depth molecular dynamics (MD) study of the behaviour of a representative probe of this family, 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphoethanolamine labelled with 3-carboxycoumarin (POPE-COUM), in bilayers of 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine (POPC)/1-stearoyl-2-linoleoyl-sn-glycero-3-phosphocholine (SLPC) 2:1, mimicking the composition of the egg yolk lecithin membranes recently used experimentally by our group to study POPE-COUM as a biomarker of the oxidation state and integrity of large unilamellar vesicles (LUVs). The MD simulations revealed that the coumarin group is oriented towards the bilayer interior, leading to a relatively internal location, in agreement with what is observed in the nitrobenzoxadiazole fluorophore of commercial head-labelled NBD-PE probes. This behaviour is consistent with the previously stated hypothesis that POPE-COUM is entirely located within the LUVs structure. Hence, the delay on the oxidation of the probe in the oxygen radical absorbance capacity (ORAC) assays performed is related with the inaccessibility of the probe until alteration of the LUV structure occurs. Furthermore, our simulations show that POPE-COUM exerts very little global and local perturbation on the host bilayer, as evaluated by key properties of the unlabelled lipids. Together, our findings establish PE-COUM as suitable fluorescent lipid analogue probes.

Design and application of Cd2+ polypeptide fluorescent probes based on Aggregation Induced Emission (AIE).

Cadmium is a toxic heavy metal, which is both an environmental pollutant, and a threat to human health. A fluorescent probe was developed to detect Cd2+ selectively, sensitively, and quickly. This study reports the successful development of a polypeptide fluorescent probe TPE-HC (TPE-His-Pro-Gly-Cys) which selectively detects Cd2+ by Aggregation-Induced Emission effect. After fluorescence excitation, Cd2+ can be effectively detected based on the change of fluorescence intensity. The detection limit of Cd2+ in buffer solution was determined to be 151 nM (R2 = 0.9933). This probe exhibits high sensitivity, high cell permeabilit y, and low biological toxicity, and can perform live cell imaging under biological conditions. This study indicates that TPE-HC can detect Cd2+ in biological environments.

Different lateral packing stress in acyl chains alters KcsA orientation and structure in lipid membranes.

The molecular structures of the various intrinsic lipids in membranes regulate lipid-protein interactions. These different lipid structures with unique volumes produce different lipid molecular packing stresses/lateral stresses in lipid membranes. Most studies examining lipid packing effects have used phosphatidylcholine and phosphatidylethanolamine (PE), which are the main phospholipids of eukaryotic cell membranes. In contrast, Gram-negative or Gram-positive bacterial membranes are composed primarily of phosphatidylglycerol (PG) and PE, and the physical and thermodynamic properties of each acyl chain in PG at the molecular level remain unresolved. In this study, we used 1-palmitoyl-2-oleoyl-sn-glycero-3-phospho-(1'-rac-glycerol) (POPG, 16:0-18:1 PG) and 1-palmitoyl-2-arachidonoyl-sn-glycero-3-phospho-(1'-rac-glycerol) (PAPG, 16:0-20:4 PG) to prepare lipid bilayers (liposome) with the rod-type fluorescence probe DPH. We measured the lipid packing conditions by determining the rotational freedom of DPH in POPG or PAPG bilayers. Furthermore, we investigated the effect of different monoacyl chains on a K+ channel (KcsA) structure when embedded in POPG or PAPG membranes. The results revealed that differences in the number of double bonds and carbon chain length in the monoacyl chain at sn-2 affected the physicochemical properties of the membrane and the structure and orientation of KcsA.

On-Site Quantitative Visualization of Singlet Oxygen in Crops via an Organic Small Molecule-Based Ratiometric Fluorescent Probe and a Mobile Fluorescence Analysis Device.

Singlet oxygen (1O2) plays imperative roles in a variety of biotic or abiotic stresses in crops. The change of its concentration within a crop is closely related to the crop growth and development. Accordingly, there is an urgent need to develop an efficient analytical method for on-site quantitative detection of 1O2 in crops. Here, we judiciously constructed a novel ratiometric fluorescent probe, SX-2, for the detection of 1O2 in crops. Upon treating with 1O2, probe SX-2 displayed highly selective ratiometric fluorescence response, which is favorable for the quantitative detection of 1O2. Concurrently, the fluorescence solution color of probe SX-2 was varied, obviously from blue to yellow, indicating that the probe is beneficial for on-site detection by the naked eye. Sensing reaction mechanism studies showed that the 2,3-diphenyl imidazole group in SX-2 could function as a new selective recognition group for 1O2. Probe SX-2 was utilized for the detection of photoirradiation-induced 1O2 and endogenous 1O2 in living cells. The changes in the 1O2 level in zebrafish were also tracked by fluorescence imaging. In addition, the production of 1O2 in crop leaves under a light source of different wavelengths was studied. The results demonstrated more 1O2 were produced under a light source of 365 nm. Furthermore, to achieve on-site quantitative detection, a mobile fluorescence analysis device has been made. Probe SX-2 and mobile fluorescence analysis device were capable of on-site quantitative detecting of 1O2 in crops. The method developed herein will be convenient for the on-site quantitative measurement of 1O2 in distinct crops.

Advances in preclinical approaches for intravesical therapy of bladder cancer.

The purpose of this review is to explore new strategies to treat bladder cancer. This article addresses challenges and opportunities in intravesical therapy of bladder cancer. The review examines the latest advances in the development of preclinical approaches for intravesical therapy of bladder cancer. It discusses strategies to improve drug delivery efficiency by using synthesized diverse carriers. Immunotherapy with protein aggregate magnesium-ammonium phospholinoleate-palmitoleate anhydride has been shown to be more effective than intravesical Bacillus Calmette-Guerin. Novel drug delivery systems such the urinary drug-disposing strategy and intravesical nanoparticle formulations improve the drug delivery efficiency while minimizing adverse reactions. Innovative imaging techniques using near-infrared fluorescence probes and multifunctional nano-transformers enable real-time detection and targeted therapy in bladder cancer treatment. Treatment of bladder cancer is clinically challenging. However, recent progress in drug delivery technologies shows promise. Optimizing these technologies helps improve patient outcomes, and facilitates clinical translation of different treatment modalities.

Green-synthesized chitosan‑carbon dot nanocomposite as turn-on aptasensor for detection and quantification of Leishmania infantum parasite.

Leishmania is one of the most common diseases between human and animals, caused by Leishmania infantum parasite. Here, we have developed an ultra-selective turn-on fluorescent probe based on an aptamer and Chitosan-CD nanocomposite. The CD used in this study were synthesized using Quercus cap extract and a microwave-assisted approach. The Chitosan-CD nanocomposite was optimized using several microscopic and spectroscopic techniques to possess a bright fluorescence emission before adding aptamer and totally quenched fluorescence after addition of aptamer. The designed probe was proficient in the detection and quantification Leishmania infantum parasite by selective targeting of poly(A) binding protein (PABP) on the surface of the parasite. The designed fluorescent biosensor with high sensitivity, excellent selectivity, and a limit of detection (LOD) of 94cells/mL of the Leishmania infantum parasite as well as a linear response in the ranges of 188-750 cells/mL and 3000-6000 cells/mL (R2 ≥ 0.98 for both linear ranges). Additionally, the selectivity of the designed probe was evaluated in the presence of different pathogenic species such as Trypanosoma brucei parasite and Staphylococcus aureus bacteria, as well as LiIF2α and LiP2a and BSA proteins as interference substances. The results of this study shows that using Chitosan-CD nanocomposite is a great strategy for developing selective turn-on probes with extraordinary accuracy and sensitivity in identifying Leishmania infantum parasite, especially in the early stages of the disease, and it is promising for the future clinical applications.

Correlative single molecule lattice light sheet imaging reveals the dynamic relationship between nucleosomes and the local chromatin environment.

In the nucleus, biological processes are driven by proteins that diffuse through and bind to a meshwork of nucleic acid polymers. To better understand this interplay, we present an imaging platform to simultaneously visualize single protein dynamics together with the local chromatin environment in live cells. Together with super-resolution imaging, new fluorescent probes, and biophysical modeling, we demonstrate that nucleosomes display differential diffusion and packing arrangements as chromatin density increases whereas the viscoelastic properties and accessibility of the interchromatin space remain constant. Perturbing nuclear functions impacts nucleosome diffusive properties in a manner that is dependent both on local chromatin density and on relative location within the nucleus. Our results support a model wherein transcription locally stabilizes nucleosomes while simultaneously allowing for the free exchange of nuclear proteins. Additionally, they reveal that nuclear heterogeneity arises from both active and passive processes and highlight the need to account for different organizational principles when modeling different chromatin environments.

A Novel Fluorescent Probe containing dihydropyrimidinone Analogue for Highly Sensitive, Selective Detection of Al(III) Ions with its Molecular Docking, ADME/T and Anticancer Impact

A Novel Fluorescent Probe containing dihydropyrimidinone Analogue for Highly Sensitive, Selective Detection of Al(III) Ions with its Molecular Docking, ADME/T and Anticancer Impact

Pentacenequinone-Modulated 2D GdSn-PQ Nanosheets as a Fluorescent Probe for the Detection of Enrofloxacin in Biological and Environmental Samples.

The fate and effects of fluoroquinolone antibacterial (FQ) on the environment are important since there appears to be a surge in FQ resistance like enrofloxacin (ENR) in both environmental and clinical organisms. Numerous reports indicate that the sensing capabilities of these antibiotics need to be improved. Here, we have investigated the interaction of ENR with our synthesized pentacenequinone-modulated gadolinium-tin (GdSn-PQ) nanosheets and the formation of intermolecular interactions that caused the occurrence of aggregation-induced emission enhancement. The concept for designing hybrid metallic nanosheets comes from the unique features inherited from the parent organic precursor. Due to the distinct interaction between ENR and GdSn-PQ, the interstate conversion (ISC) between GdSn-PQ and ENR induces a significant wavelength shift in photoluminescence (PL), improving reliability, selectivity, and visibility compared to quenching- or AIEE-based methods without peak shifts, allowing for highly sensitive and visually detectable analyses. The fluorescence signal of GdSn-PQ exhibited a linear relationship (R2 = 0.9911), with the added ENR concentrations ranging from 5 to 90 nM, with a detection limit of 0.10 nM. We have demonstrated its potential and wide use in the detection of ENR in biological samples (human urine and blood serum) and environmental samples (tap water and seawater) with a recovery rate of 98- 108%. The current approach has demonstrated that the 2D GdSn-PQ nanosheet is a novel and powerful platform for future biological and environmental studies.

Synthesis and application of a novel fluorescent probe based on benzothiazole for detection of hydrazine in real samples

Synthesis and application of a novel fluorescent probe based on benzothiazole for detection of hydrazine in real samples

An Imidazo[1, 2-a]Pyridine Based Fluorescent Probe for the Continuous "on-off-on" Detection of Fe3+ and F.

An "on-off-on" fluorescent probe LK was synthesized from 2-benzoylpyridine and o-vanillin, which could sequentially detect Fe3+ and F- in DMSO/H2O solutions (v/v = 1/1, HEPES buffer, 1.0 mM, pH 7.0) with large Stokes shift (178nm). LK exhibited not only high selectivity and sensitivity towards Fe3+ and F-, but also strong anti-interference ability to other ions. LK was coordinated with Fe3+ at a ratio of 2:1, with a binding constant (Ka) of 1.3 × 104 M- 1. The detection limits for Fe3+ and F- were 6.9 × 10- 8 M and 3.0 × 10- 7 M, respectively. Due to its excellent sensing performance, LK was successfully applied in actual water samples and test papers for the detection of Fe3+ and F-.

The extract of buds of Chrysanthemum morifolium Ramat alleviated UVB-induced skin photoaging by regulating MAPK and Nrf2/ARE pathways.

Chrysanthemum morifolium Ramat. is a commonly used Chinese herb and food homologous plant with traditional effects such as anti-inflammatory, antifebrile, antibacterial and antiviral. Photoaging is one of the main causes of accelerated skin aging. Chrysanthemum morifolium Ramat. has reported to alleviate photodamage. In this study, we investigated the protective effect of the extract of buds of Chrysanthemum morifolium Ramat. (CE) on UVB-induced photoaging and further mechanism. The extract of buds of chrysanthemum was analyzed by HPLC-Q-TOF-MS/MS. Antioxidant activity was assessed by DPPH and ABTS assay. Cell viability examined by cell counting kit-8 assay. The ROS level was detected by fluorescent probe DCFH-DA. Protein expression evaluated by Western blotting. The skin tissue investigated by immunohistochemistry. CE significantly reversed the decrease of cell viability that induced by UVB in HaCaT and HFF-1 cells. Further analysis showed that CE alleviated photoaging by inhibiting the expression of mitogen-activated protein kinase (MAPK) and activating the NF-E2-related factor 2 (Nrf2)/ antioxidant response element (ARE) pathway to promote the expression of antioxidant enzymes. Moreover, CE effectively improved the reduced skin hydration, disordered collagen and thickening epidermis caused by UVB in mice. All results demonstrated that CE had therapeutic effect on UVB-induced photoaging and provided theoretical basis for its further developing as a natural functional product with anti-photoaging effect.

High-Affinity NIR-Fluorescent Inhibitors for Tumor Imaging via Carbonic Anhydrase IX.

Tumor imaging and delivery of therapeutic agents may be achieved by designing high-affinity and high-selectivity compounds recognizing a tumor cell-expressing biomarker, such as carbonic anhydrase IX (CA IX). The CAIX, overexpressed in many hypoxic solid tumors, helps adjust to the energy requirements of the hypoxic environment, reduces intracellular acidification, and participates in the metastatic invasion of adjacent tissues. Here, we designed a series of sulfonamide compounds bearing CAIX-recognizing, high-affinity, and high-selectivity groups conjugated via a PEG linker to near-infrared (NIR) fluorescent probes used in the clinic for optically guided cancer surgery. We determined compound affinities for CAIX and other 11 catalytically active CA isozymes by the thermal shift assay and showed that the affinity Kd value of CAIX was in the subnanomolar range, hundred to thousand-fold higher than those of other CA isozymes. Similar affinities were also observed for CAIX expressed on the cancer cell surface in live HeLa cell cultures, as determined by the competition assay. The NIR-fluorescent compounds showed excellent properties in visualizing CAIX-positive tumors but not CAIX-negative knockout tumors in a nude mice xenograft model. These compounds would therefore be helpful in optically guided cancer surgery and could potentially be developed for anticancer treatment by radiotherapy.

Bora-diaza-indacene based fluorescent probes for simultaneous visualisation of lipid droplets and endoplasmic reticulum.

Organelle selective fluorescent probes, especially those capable of concurrent detection of specific organelles, are of benefit to the research community in delineating the interplay between various organelles and the impact of such interaction in maintaining cellular homeostasis and its disruption in the diseased state. Although very useful, such probes are synthetically challenging to design due to the stringent lipophilicity requirement posed by different organelles, and hence, the lack of such probes being reported so far. This work details the synthesis, photophysical properties, and cellular imaging studies of two bora-diaza-indacene based fluorescent probes that can specifically and simultaneously visualise lipid droplets and endoplasmic reticulum; two organelles suggested having close interactions and implicated in stress-induced cellular dysfunction and disease progression.

Intrabacterial Nitroreductase‐Activated Imaging and Persistently Illuminated Photodynamic Therapy for Intracellular Infection Theranostics

AbstractIntracellular bacterial (ICB) infection is one of the most life‐threatening cases of drug resistance, and photodynamic therapy (PDT) is challenged by limited light penetration into tissues and unspecific toxicity to host cells. Herein, theranostic nanoparticles (NPs) are developed to facilitate on‐demand activation of photosensitizers inside ICBs and persistent PDT of deep‐tissue ICB infections. Mechanoluminescent SrAl2O4:Eu2+ and persistence luminescent CaS:Eu2+ are sequentially deposited on mesoporous silica to prepare mSC, followed by encapsulation by bacterial membranes with loaded nitroreductase (NTR)‐activatable methylene blue (nMB) to obtain mSC‐nMB@BM NPs. The fluorescence quenching of nMB with mSC offers few cytotoxicities even under ultrasonication, and bacterial membrane coatings mediate the specific NP internalization into ICB‐infected macrophages. Bacterial pore‐forming toxins trigger nMB release from NPs, and intrabacterial NTR rejuvenates methylene blue probes from nMB for real‐time imaging of ICB infections. Ultrasonication of NPs continuously excites the rejuvenated methylene blue to generate reactive oxidative species and reduce viable ICBs by 3.54 log magnitude folds. NP treatment on ICB‐infected mice exhibits full survival and restores viscera functions without any pathological abnormality. Therefore, this concise NP design demonstrates capabilities of bacterial membrane‐mediated specific internalization, bacterial toxin‐triggered release and intrabacterial NTR‐rejuvenation of fluorescent probes, and persistent internal light‐illumination for ICB theranostics.

A new three-dimensional (3D) molecularly imprinted polymer fluoroprobe based on green-red dual-emission signals of carbon quantum dots and self-polymerization of dopamine (CDs@PDA-MIPs) for sensitive detection of nifedipine.

Nifedipine (NIF), as one of the dihydropyridine calcium channel blockers, is widely used in the treatment of hypertension. However, misuse or ingestion of NIF can result in serious health issues such as myocardial infarction, arrhythmia, stroke, and even death. It is essential to design a reliable and sensitive detection method to monitor NIF. In this work, an innovative molecularly imprinted polymer dual-emission fluorescent sensor (CDs@PDA-MIPs) strategy was successfully designed for sensitive detection of NIF. The fluorescent intensity of the probe decreased with increasing NIF concentration, showing a satisfactory linear relationship within the range 1.0 × 10-6M ~ 5.0 × 10-3M. The LOD of NIF was 9.38 × 10-7M (S/N = 3) in fluorescence detection. Theapplication of the CDs@PDA-MIPs in actual samples such as urine and Qiangli Dingxuan tablets has been verified, with recovery ranging from 97.8 to 102.8% for NIF. Therefore, the fluorescent probe demonstrates great potential as a sensing system for detecting NIF.

Activity-based protein profiling of serine hydrolases and penicillin-binding proteins in Enterococcus faecium

Abstract Enterococcus faecium is a gut commensal bacterium that is gaining increasing relevance as an opportunistic, nosocomial pathogen. Its high level of intrinsic and acquired antimicrobial resistance is causing a lack of treatment options, particularly for infections with vancomycin-resistant strains, and prioritizes the identification and functional validation of novel druggable targets. Here, we use activity-based protein profiling (ABPP), a chemoproteomics approach using functionalized covalent inhibitors, to detect active serine hydrolases across 11 E. faecium and Enterococcus lactis strains. Serine hydrolases are a big and diverse enzyme family, that includes known drug targets such as penicillin-binding proteins (PBPs), whereas other subfamilies are underexplored. Comparative gel-based ABPP using Bocillin-FL revealed strain- and growth condition dependent variations in PBP activities. Profiling with the broadly serine hydrolase-reactive fluorescent probe fluorophosphonate-TMR showed a high similarity across E. faecium clade A1 strains, but higher variation across A2 and E. lactis strains. To identify these serine hydrolases, we used a biotinylated probe analog allowing for enrichment and identification via liquid chromatography-mass spectrometry. We identified 11 largely uncharacterized targets (α,β-hydrolases, SGNH-hydrolases, phospholipases, amidases, peptidases) that are druggable and accessible in live vancomycin-resistant E. faecium E745 and may possess vital functions that are to be characterized in future studies.