Fluorescent Probe For Imaging Research Articles

A protein structure-dependent fluorescent probe for hemoglobin monitoring and controllable imaging in living cells.

A novel protein structure-dependent non-covalent fluorescent probe, DDBM, was developed. It exhibited selective fluorescence "turn-off" responsiveness to bovine hemoglobin (BHb). This responsiveness depended on the interaction between the probe and BHb, with the methoxynaphthalene group significantly contributing to the sensitivity. Non-covalent interactions played a crucial role in stabilizing the binding of DDBM with BHb. DDBM demonstrated a robust anti-interference capability in its BHb responsiveness. Interestingly, the BHb responsiveness of DDBM could be modulated by ibuprofen. Additionally, DDBM exhibited favorable fluorescence enhancement sensitivity to bovine serum albumin (BSA), coupled with a robust anti-interference capability. These distinctive properties of DDBM enabled it to dynamically trace the metabolism of hemoglobin (Hb) and further achieve Hb-mediated precise controllable live cell imaging.

Endogenous Telomerase-Activated Fluorescent Probes for Specific Detection and Imaging of Flap Endonuclease 1 in Cancer Cells and Tissues.

Flap endonuclease 1 (FEN1) is a structure-specific DNA repair enzyme that has emerged as a potential target for cancer diagnosis and treatment. However, existing FEN1 assays often suffer from complicated reaction schemes and laborious procedures, and only a few methods are available for the detection and imaging of FEN1 in living cells. Especially, FEN1 is not exclusive to cancer cells, but it is also shared by normal cells. Consequently, the specific detection of FEN1 in cancer cells remains a challenge. Herein, we develop a simple and selective fluorescent biosensor for the specific imaging of FEN1 in cancer cells and tissues by engineering a FEN1 detection probe with a telomerase-responsive unit. In the presence of telomerase, it induces an extension reaction and subsequent intramolecular reconfiguration of the detection probe, generating a suitable branched DNA structure for FEN1 recognition and facilitating the cleavage of the flap by FEN1 for the recovery of fluorescence signal. Because telomerase is undetectable in normal cells but highly upregulated in cancer cells, the detection probe can only be activated in cancer cells to generate a high signal. This assay is quite simple, with the requirement of merely a single probe for dual enzyme recognition and signal output. With the integration of the single-molecule counting technology, this biosensor can achieve a detection limit of 1.2 × 10-5 U/μL, and it can accurately detect FEN1 in living cells and clinical tissues, providing a new avenue for FEN1-associated fundamental research and clinical diagnosis.

A Near-Infrared Fluorescent Probe for Live-Cell Imaging of RNA in the Cancer Cells.

Ribonucleic acid (RNA), a biomolecular compound polymerized by many riboside monomers, is one of the most essential substances in life. Monitoring RNA changes within organisms will play an important role in the diagnosis and treatment of major genetically related diseases. Herein, we synthesized a novel compound QOT-NA as near-infrared probe for imaging RNA in cancer cells. The synthesis method and photophysical properties of the probe were presented. The titration and selectivity experiments demonstrated that the probe detected RNA in vitro. Moreover, the RNA imaging experiments indicated that the near-infrared RNA probe QOT-NA can locate RNA within cancer cells.

Water-Soluble Bimodal Magnetic-Fluorescent Radical Dendrimers as Potential MRI-FI Imaging Probes.

Dual or multimodal imaging probes have become potent tools for enhancing detection sensitivity and accuracy in disease diagnosis. In this context, we present a bimodal imaging dendrimer-based structure that integrates magnetic and fluorescent imaging probes for potential applications in magnetic resonance imaging and fluorescence imaging. It stands out as one of the rare examples where bimodal imaging probes use organic radicals as the magnetic source, despite their tendency to entirely quench fluorophore fluorescence. Opting for organic radicals over metal-based contrast agents like gadolinium (Gd3+)-chelates is crucial to mitigate associated toxicity concerns. We utilized an amino-terminated polyamide dendrimer containing a 1,8-naphthalimide (Naft) fluorescent group, amino acid derivatives as linkers to enhance water solubility, and TEMPO organic radicals as terminal groups. The same dendrimer structure, featuring an equivalent number of branches but lacking the fluorophore group, was also functionalized with amino acid and terminal radicals to serve as a reference. Remarkably, we achieved a fully water-soluble dendrimer-based structure exhibiting both magnetic and fluorescent properties simultaneously. The fluorescence of the Naft group in the final structure is somewhat quenched by the organic radicals, likely due to photoinduced electron transfer with the nitroxyl radical acting as an electron acceptor, which has been supported by density functional theory calculations. Molecular dynamics simulations are employed to investigate how the dendrimers' structure influences the electron paramagnetic resonance characteristics, relaxivity, and fluorescence. In summary, despite the influence of the radicals-fluorophore interactions on fluorescence, this bimodal dendrimer demonstrates significant fluorescent properties and effective r1 relaxivity of 1.3 mM-1 s-1. These properties have proven effective in staining the live mesenchymal stem cells without affecting the cell nucleus.

Balancing Brightness and Photobasicity: Modulating Excited-State Proton Transfer Pathways in Push-Pull Fluorophores for Biological Two-Photon Imaging.

Push-pull fluorophores with donor-π-acceptor architectures are attractive scaffolds for the design of probes and labels for two-photon microscopy. Such fluorophores undergo a significant charge-delocalization in the excited state, which is essential for achieving a large two-photon absorption cross-section and brightness. The polarized excited state may, however, also facilitate excited-state proton transfer (ESPT) pathways that can interfere with the probe response. Herein, we employed steady-state and time-resolved spectroscopic studies to elucidate whether ESPT is responsible for the pH-dependent emission response of the Zn(II)-selective fluorescent probe chromis-1. Composed of a push-pull architecture with a pyridine ring as the acceptor, the chromis-1 fluorophore core acts as a photobase that promotes ESPT upon acidification. Although the pKa of the pyridine acceptor increases more than six orders of magnitude upon excitation, the photobasicity is not sufficient to deprotonate solvent water molecules under neutral conditions. Rather, the pH-dependent emission response is caused by the pendant bis-isonicotinic acid chelating group which upon protonation facilitates an excited-state intramolecular proton transfer to the pyridine acceptor. A simple permutation of the core pyridine nitrogen from the para- to the ortho-position relative to the thiazole substituent was sufficient to reduce the excited-state basicity by two orders of magnitude without compromising the two-photon excited brightness. These results highlight the importance of choosing the appropriate fluorophore core and chelating moiety for minimizing pH-dependent responses in the design of fluorescent probes for biological imaging.

Cellulosic schiff base hydrogel biosensor for bacterial detection with pH/thermo-responsitivity: DFT calculations and molecular docking

A facile green hydrothermal method was developed for the preparation of fluorescent carbon dots (CDs) using sugarcane bagasse (SB) as a raw material. The incorporation of CDs within the hydroxyethyl cellulose-acrylamide hydrogel (HEC-AM) in the presence of glutardehyde (GA) as a crosslinking agent showed bright red emission under fluorescence microscope. The hydroxyethyl cellulose-N–CDs-acrylamide hydrogel (HEC-AM@N–CDs) were used as alternative biocompatible fluorescent probes for imaging and suppression of Gram-negative bacteria such as Escherichia coli (with inhibition zones 13 mm for HEC-AM to 17 mm for HEC-AM@N–CDs) &Helicobacter pylori (with inhibition zones 16 mm for HEC-AM to 13 mm for HEC-AM@N–CDs) and Gram-positive bacteria such as Micrococcus luteus (with inhibition zones 15 mm for HEC-AM to 16 mm for HEC-AM@N–CDs) &Staphylococcus aureus (with inhibition zones 13 mm for HEC-AM to 14 mm for HEC-AM@N–CDs). The N–CDs promote bacterial binding and internalization, leading to differential fluorescence emission depending on the bacteria's cell wall composition. DFT calculations support strong interactions between the bacteria and HEC-AM@N–CDs.

Clog P-Guided Development of Multi-Colored Buffering Fluorescent Probes for Super-Resolution Imaging of Lipid Droplet Dynamics.

Super-resolution fluorescence imaging of live cells increasingly demands fluorescent probes capable of multi-color and long-term dynamic imaging. Understanding the mechanisms of probe-target recognition is essential for the engineered development of such probes. In this study, it is discovered that the molecular lipid solubility parameter, Clog P, determines the staining performance of fluorescent dyes on lipid droplets (LDs). Fluorescent dyes with Clog P values between 2.5 and 4 can form buffering pools outside LDs, replacing photobleached dyes within LDs to maintain constant fluorescence intensity in LDs, thereby enabling dynamic super-resolution imaging of LDs. Guided by Clog P, four different colored buffering LD probes spanning the visible light spectrum have been developed. Using Structured Illumination Microscopy (SIM), the role of LD dynamics have been tracked during cellular ferroptosis with the secretion, storage, and degradation of overexpressed ACSL3 proteins. It is found that LDs serve as storage sites for these proteins through membrane fusion, and further degrade overexpressed proteins via interactions with organelles like lysosomes or through lipophagy, thereby maintaining cellular homeostasis.

Sensing and Imaging Agents for Cyclooxygenase Enzyme.

In this concept, we present a comprehensive study on the development and application of COX-2-specific fluorescent probes for cancer imaging and diagnosis. Targetting cancer cells and measuring cancer-related activities in specific organelles quickly and accurately are crucial factors for early diagnosis and research on cancer pathology and treatment. This concept explores a variety of probes which include BDIMC1-2, TPI-IMC, YZP1, IQ-1, ANQ-IMC-6, Niblue-C6-IMC, AZB-IMC2, CMP, CF3-Fluorocoxib, IBPC1, CoxFluor, etc. and each one demonstrates unique mechanisms and high selectivity towards COX-2 enzymes. These probes were designed to enhance fluorescence upon binding to COX-2 which enable precise visualization of tumor and inflamed tissues. The research emphasizes the importance of COX-2 as a biomarker in cancer diagnostics, particularly in identifying cancer stem cells and inflamed tissues. This concept highlights the potentiality of these probes in non-invasive imaging techniques which offering significant advancements in cancer diagnosis and monitoring. The in vivo and in vitro experiments, including applications in mouse models and human tissue samples, confirm the efficacy of these probes in providing detailed imaging for clinical and research applications.

Multivalent supramolecular fluorescent probes for accurate disease imaging.

Optical imaging is a powerful tool for early disease detection and effective treatment planning, but its accuracy is often compromised by the uptake of imaging materials by the mononuclear phagocyte system (MPS). Herein, we leverage multivalent host-guest interactions between cyanine dyes and β-cyclodextrin polymers to develop supramolecular probes with enhanced stability, optical, and transport profiles for accurate in vivo imaging. These multivalent interactions not only ensure the stability of the probes but also enhance fluorescence efficiency by minimizing nonradiative decay. Our self-assembly approach effectively modulates probe size and surface properties, enabling evasion of MPS clearance and promoting prolonged bloodstream circulation, thereby improving the signal-to-background ratio for imaging. The effectiveness of our design is demonstrated by substantial advancements in the early diagnosis of acute kidney injury and by providing high-contrast imaging and precise surgical navigation across various tumor models. Our strategy not only advances optical imaging materials toward clinical translation but also establishes a versatile platform applicable to multiple imaging modalities.

An intramolecular charge transfer based fluorescent probe for imaging of OCl–

The discovery and utilization of new fluorescent chromophore is indispensable to exploit high performance probes for biological research. Stokes shift is one of the most important properties of chromophore accounting for super-resolution fluorescence imaging. Intramolecular charge transfer (ICT) is one of the fundamental mechanisms for fluorescence that accompanied by large Stokes shifts. Based on the conformational changes between ground and excited states, ICT models can be divided into two types: conformation-steady ICT, whose conformation remains unchanged, and conformation-changeable ICT, which is characterized by the rotation of the chromophore around an axis upon excitation. Herein, we report a new chromophore whose donor and acceptor parts took a butterfly geometry with a dihedral angle of 21° in ground state and a planar conformation upon photo excitation. The bent conformation might be ascribed to the extra conjugated double bond, which made the coplanarity of the chromophore in ground state get worse. The chromophore shows a remarkable Stokes shift over 150 nm and a high fluorescence quantum yieldof 0.62. The limit of detection is 41 nM, which enabled the imaging of basal as well as induced OCl– in different cells. Moreover, the pronounced spectroscopic properties ensure the in vivo monitoring of OCl– in arthritic mice. This finding would shed light on the exploitation of small molecule probes based on new fluorescence chromophore for precise biological imaging.

Selective Recognition of the Dimeric NG16 Parallel G-Quadruplex Structure Using Synthetic Turn-On Red Fluorescent Protein Chromophore.

Red fluorescent protein (RFP)-based fluorescent probes that can selectively interact with specific nucleic acids are of great importance for therapeutic and bioimaging applications. Herein, we have reported the synthesis of RFP chromophores for selective recognition of G-quadruplex nucleic acids in vitro and ex vivo. We identified DFHBI-DM as a fluorescent turn-on probe that binds to the dimeric NG16 parallel quadruplex with superior selectivity and sensitivity over various parallel, antiparallel, and hybrid topologies. The binding of DFHBI-DM to NG16 exhibited excellent photophysical properties, including high binding affinity, large Stokes shift, high photostability, and quantum yield. The MD simulation study supports the 1:1 binding stoichiometry. It confirms the planar conformation of DFHBI-DM, which makes strong binding interactions with a flat quartet of NG16 compared to other antiparallel and hybrid topologies. The cell imaging and MTT assays revealed that DFHBI-DM is a biocompatible and efficient fluorescent probe for intracellular imaging of NG16. Overall, these results demonstrated that DFHBI-DM could be an effective fluorescent G4-stabilizing agent for the dimeric NG16 parallel quadruplex, and it could be a promising candidate for further exploration of bioimaging and therapeutic applications.

Water-soluble sulfur quantum dots as a potential sensitive fluorescent probe for quercetin detection and cell imaging

A simple fluorescent quenching probe based on polyethylene glycol-400 capped sulfur quantum dots (PEG-SQDs) was fabricated to determine quercetin (QT) quantitatively. As anticipated, the PEG-SQDs exhibited favourable luminescent properties, stability and low cytotoxicity. QT effectively quenched the fluorescence of the PEG-SQDs through static quenching and the inner filter effect. Moreover, the PEG-SQDs showed rapid QT detection within a linear range of 0.100–45.0 μM, with a limit of detection of 0.014 μM (3σ/k). This fluorescent probe successfully detected QT in human serum, quercetin supplement capsules and red wine, achieving a standard recovery of 92.6 %–105 %.

Novel anti-CEA affibody for rapid tumor-targeting and molecular imaging diagnosis in mice bearing gastrointestinal cancer cell lines.

Gastrointestinal cancer is a common malignant tumor with a high incidence worldwide. Despite continuous improvements in diagnosis and treatment strategies, the overall prognosis of gastrointestinal tumors remains poor. Carcinoembryonic antigen (CEA) is highly expressed in various types of cancers, especially in gastrointestinal cancers, making it a potential target for therapeutic intervention. Therefore, the expression of CEA can be used as an indication of the existence of tumors, chosen as a target for molecular imaging diagnosis, and effectively utilized in the targeted therapy of gastrointestinal cancers. In this study, we report the selection and characterization of affibody molecules (ZCEA539, ZCEA546, and ZCEA919) specific to the CEA protein. Their ability to bind to recombinant and native CEA protein has been confirmed by surface plasmon resonance (SPR), immunofluorescence, and immunohistochemistry assays. Furthermore, Dylight755-labeled ZCEA affibody showed accumulation within the tumor site 1 h post injection and was continuously enhanced for 4 h. The Dylight755-labeled ZCEA affibody exhibited high tumor-targeting specificity in CEA+ xenograft-bearing mice and possesses promising characteristics for tumor-targeting imaging. Overall, our results suggest the potential use of ZCEA affibodies as fluorescent molecular imaging probes for detecting CEA expression in gastrointestinal cancer.

Excitation energy transfer based naphthalimide-boron-dipyrromethene dyads as two-photon fluorescent probes for palladium(0) detection and live cell imaging

Increasing industrial demand has accelerated the consumption and contamination of palladium (Pd) worldwide. The implementation of fluorescent probes, especially with two-photon excitation, holds great promise for Pd detection in environmental and biological analysis. In this work, two naphthalimide (NPI)-boron-dipyrromethene (BDP) dyads (named NBH and NBN) were developed as two-photon fluorescent probes for Pd(0) detection based on an excitation energy transfer (EET) strategy, employing NPI as the energy donor and BDP as the acceptor. Spectroscopic studies revealed that both probes exhibited a fast response to Pd(0) in a pronounced fluorescence "ON-OFF" manner. The Pd-catalyzed Tsuji-Trost reaction activated an intramolecular photoinduced electron transfer (PET) process between the aniline and BDP moieties, resulting in the fluorescence quenching of the BDP fluorophore. The reaction-based probes exhibited excellent sensitivity and selectivity for Pd(0) species with a detection limit as low as 2.4 nM. These probes were successfully applied to the determination of Pd residues in drug, soil, and water samples. A simple smartphone-based platform was also constructed to determine Pd(0) via an RGB reader. With negligible cytotoxicity, confocal imaging study validated their feasibility of monitoring Pd(0) in living cells through multiple excitation channels.

The Influence of the Functional Group on the Physicochemical and Biological Properties of New Phenanthro[9,10-d]-Imidazole Derivatives.

The search for safe, cheap, and repeatable diagnostic methods is a fundamental research goal. Currently, great hope is placed on fluorescence imaging. However, the development of this method mainly depends on efficient fluorescent probes. Designing and obtaining new probes with potential applications in fluorescence imaging is very difficult because compounds of this type must meet several requirements related to their properties. Therefore, this article attempted to obtain and study new phenanthro[9,10-d]-imidazole derivatives (PK1-PK3) with potential application as fluorescent probes for fluorescence imaging. The main goal of the work was to assess the effect of two functional groups (such as the formyl group (PK2) and rhodanine-3-acetic acid (PK3)) on selected physicochemical properties and possibilities of practical application of the considered compounds. The conducted studies proved that the influence of the functional group is significant, as it causes a bathochromic shift in both absorption and emission results (by the order PK1 < PK2 < PK3). Moreover, all compounds could stain live cells cultured in vitro. The staining efficiency was not affected by the cell line, thanks to which we obtained the correct staining of both mouse and human cell lines. PK3 was the most attractive of the tested compounds due to its staining potential of live cells and retention after fixation. Our results also showed some antibacterial and antifungal activity of the newly synthesized compounds (PK1-PK3). Among them, PK3 showed the highest antimicrobial effect, especially against Gram-positive bacteria.

Renal-Clearable Organic Probes From D-A-D Type Aza-BODIPY Fluorophores for Multiphoton Deep-Brain Imaging.

Bright near-infrared (NIR) fluorescent probes play an important role in in vivo optical imaging. Here, renal-clearable nanodots prepared from Aza-BODIPY are reported fluorophores for multiphoton brain imaging. The design of donor-acceptor-donor (D-A-D) type conjugated structures endowed the fluorophores with large three-photon absorption cross-section for both 1620 and 2200nm excitation. The side chain modification and lipid encapsulation yield ultrasmall nanodots (≈4nm) and a high fluorescence quantum yield (≈0.35) at 720nm emission in the aqueous phase. The measured three-photon action cross-section of a single Aza-BODIPY fluorophore in the nanodots is ≈30 times higher than the commonly used Sulforhodamine 101 dye. Three-photon deep brain imaging of subcortical structures is demonstrated, reaching a depth of 1900µm below the brain surface in a live mouse study. The nanodots enabled blood flow measurement at a depth of 1617µm using line scanning three-photon microscopy (3PM). This work provides superior fluorescent probes for multiphoton deep-brain imaging.

A dual-responsive fluorescent probe for cellular hypoxia and tumor imaging based on nitroreductase and viscosity synergy

Nitroreductase (NTR), an enzyme that operates within the cytoplasm, relies on flavin mononucleotide or flavin adenine dinucleotide for its activity. In tumor cells, hypoxic conditions often lead to an increase in NTR levels. Consequently, the development of fluorescent probes capable of detecting NTR activity is crucial for the monitoring of hypoxia in biological systems. This study presents the one-step synthesis of a near-infrared fluorescent probe, DSM-NO2. The probe was highly sensitive to NTR and caused a fluorescence enhancement by 50-fold, with a detection limit as low as 1 ng/mL. Under hypoxic conditions in tumor cells, not only does NTR increase, but the cellular viscosity also rises. In high-viscosity environments, the fluorescence enhancement could reach up to 220-fold, and the detection limit could be further reduced to 55 pg/mL. The reaction mechanism was thoroughly investigated using mass spectrometry, molecular docking, and theoretical calculations. Moreover, the probe was successfully applied in the real-time monitoring and visualization analysis of NTR during the growth of Escherichia coli and in the hypoxic state of cancer cells. Additionally, through nebulized inhalation, the probe was successfully employed for in situ fluorescent imaging studies in a murine model of lung cancer.

Molecular engineering of BODIPY-bridged fluorescent probes for lysosome imaging - a computational study.

Lysosome imaging plays an important role in diagnosing many diseases and understanding various intracellular processes. Recently, B0 was reported as a fluorescent probe capable of detecting lysosomal viscosity changes. BODIPY is fused into the molecule as a bridge between the acceptor and donor components of B0, yielding nine new B molecules. Computational design and analysis of their optoelectronic properties were conducted to evaluate their effectiveness as fluorescent probes for lysosome imaging, with a specific target of HSA inside lysosomes. Optimized geometries reveal excellent π electron delocalization, resulting in nearly planar molecular structures. Frontier molecular orbital analysis suggests intramolecular charge transfer, along with π-π* transitions, from donor to bridge. TD-DFT calculations were performed to study absorption properties in the solvent phase, with B3PW91 showing good agreement with experiments. Molecular docking studies indicate that B derivatives can bind with HSA, and molecular dynamics simulations confirm their HSA targeting ability. This investigation highlights the introduction of BODIPY as a bridge for developing new probes capable of producing NIR fluorescence for bio-imaging, aiding in disease diagnosis.

Dual-responsive two-photon probe for specific lipid droplets near-infrared fluorescence imaging in the brain of epileptic mice

Abnormal lipid metabolism in glial cells is a key pathological feature of epilepsy. The identification of lipid droplets (LDs) is essential for investigating lipid metabolism, disease progression, and potential therapeutic interventions. Two-photon imaging technology enables real-time visualization of the spatial distribution and temporal dynamics of LDs in epilepsy models. In this study, we developed a novel two-photon excited dual-responsive near-infrared fluorescent probe, CabA, based on viscosity and polarity, to monitor dynamic changes in LDs. The fluorescence of CabA at 670 nm exhibits a significant increase in response to low polarity and high viscosity due to the twisted intramolecular charge transfer and intramolecular charge transfer mechanisms. The LDs-targeting capability of CabA at the cellular level and the process of LDs generation between neurons and astrocytes during the pathological advancement of epilepsy have been validated. In situ synchronous imaging experiments in epileptic and normal mice using CabA revealed abnormal LDs accumulation in the brain during seizures. Two-photon fluorescence imaging further demonstrated LDs accumulation in the brains of epileptic mice at a penetration depth of 100 μm. This study offers a valuable tool for enhancing the understanding of LDs in physiological and pathological processes, potentially aiding in the early diagnosis of epilepsy.

Catalyst Free Synthesis of 4‐Aryl/Alkylamino‐1,2‐Naphthoquinones: Applications in Fluorescence Microscopy

AbstractAn efficient one‐step reagent‐free synthesis of 4‐Aryl/alkylamino‐1,2‐naphthoquinones in water and iPrOH (1 : 1) mixture was carried out at room temperature (RT) using 4‐amino‐3‐hydroxynaphthalene‐1‐sulfonic acid and various primary and secondary aryl and alkyl amines. The reaction did not proceed in the absence of air or oxygen. In all cases, good yields (isolated yield: ≥65 %) were observed. The compounds exhibited fluorescence emission bands (400–470 nm) in the visible region upon excitation at their respective λmax values (330–350 nm). We evaluated in vitro cytotoxicity of some of the derivatives against two human breast cancer cell lines (MCF‐7, and MDA‐MB‐231) using the WST‐1 assay. The naphthoquinone derivatives did not show any cytotoxic effects at concentration levels relevant to cellular fluorescence imaging. The imaging experiments revealed that a few of the derivatives hold the potential to be used as cellular fluorescent imaging probes.