Multifunctional Probe Research Articles

Recent Development of Advanced Fluorescent Molecular Probes for Organelle-Targeted Cell Imaging.

Fluorescent molecular probes are very powerful tools that have been generally applied in cell imaging in the research fields of biology, pathology, pharmacology, biochemistry, and medical science. In the last couple of decades, numerous molecular probes endowed with high specificity to particular organelles have been designed to illustrate intracellular images in more detail at the subcellular level. Nowadays, the development of cell biology has enabled the investigation process to go deeply into cells, even at the molecular level. Therefore, probes that can sketch a particular organelle's location while responding to certain parameters to evaluate intracellular bioprocesses are under urgent demand. It is significant to understand the basic ideas of organelle properties, as well as the vital substances related to each unique organelle, for the design of probes with high specificity and efficiency. In this review, we summarize representative multifunctional fluorescent molecular probes developed in the last decade. We focus on probes that can specially target nuclei, mitochondria, endoplasmic reticulums, and lysosomes. In each section, we first briefly introduce the significance and properties of different organelles. We then discuss how probes are designed to make them highly organelle-specific. Finally, we also consider how probes are constructed to endow them with additional functions to recognize particular physical/chemical signals of targeted organelles. Moreover, a perspective on the challenges in future applications of highly specific molecular probes in cell imaging is also proposed. We hope that this review can provide researchers with additional conceptual information about developing probes for cell imaging, assisting scientists interested in molecular biology, cell biology, and biochemistry to accelerate their scientific studies.

Highly fluorescent hydroxyl groups functionalized graphitic carbon nitride for ultrasensitive and selective determination of mercury ions in water and fish samples

Heavy metal ion pollution is always a serious problem worldwide. Therefore, monitoring heavy metal ions in environmental water is a crucial and difficult step to ensure the safety of people and the environment. A mercury ion (Hg2+) fluorescence probe with excellent sensitivity and selectivity is described here. The functionalized graphitic carbon nitride nanosheets (T/G-C3N4) fluorescence probe was fabricated using melamine as a precursor by the pyrolysis technique, followed by a rapid KOH heat treatment method for 2 min. The chemical structure and morphology of the T/G-C3N4 probe were characterized using multiple analytical techniques including UV–Vis, SEM, XPS, XRD, and fluorometer spectroscopy. Geometry optimization of T/G-C3N4 as a modified probe was performed to assess its stability and interaction ability with Hg(II) via using the density function approach. The T/G-C3N4 probe showed a linear response based on quenching over the range 0–1.25 × 103 nM Hg(II); the detection limit was 27 nM. The remarkable sensitivity of T/G-C3N4 towards the Hg2+ ions was explained by the intense coordination and fast chelation kinetics of Hg2+ with the NH2, CN, C=N, and OH groups of T/G-C3N4 nanoprobe. The T/G-C3N4 probe demonstrates exceptional selectivity for Hg2+ ions among other metal ions including (Na+, Ag+, Mg2+, Fe2+, Fe3+, Co2+, Ni2+, Cd2+, K+, Ca2+, Cu2+, Pb2+, Mn2+ and Hg2+) and over a broad pH range (6–10), together with remarkable long-term fluorescence stability in water (> 30 days) and minimal toxicity. T/G-C3N4 was used to detect and quantify Hg2+ ions in tuna and mackerel fish and the results compared to ICP-AES. The results obtained offer a new simple and green technique for the design of multifunctional fluorescent probe appropriate for environmental applications.Graphical

Conspectus on nanodiagnostics as an incipient platform for detection of oral potentially malignant disorders and oral squamous cell carcinoma.

Conspectus on nanodiagnostics as an incipient platform for detection of oral potentially malignant disorders and oral squamous cell carcinoma.

A novel ratiometric fluorescence probe for the detection of copper (II) and silver(I) based on assembling dye-doped silica core-shell nanoparticles with gold nanoclusters.

Acreatively designed and constructed a multifunctional ratiometric fluorescence probe is reportedby assembling glutathione (GSH)-protected gold nanoclusters (AuNCs) with fluorescein-doped mesoporous silica nanoparticle (FS) for the detection of Cu2+ and Ag+ ions, which could eliminate most interferences by self-calibration. Under the excitation at 450 nm, the fluorescence connected with AuNCs can rapidly respond by quenching or enhancement, respectively, for Cu2+ and Ag+ ions, while the fluorescein isothiocyante (FITC) fluorescence served as reference with negligible change. The fluorescence intensity ratio showed good linear relationships with Cu2+ and Ag+ concentrations in the range 0.5-10 μM and 0.1-8 μM, respectively. The detection limits were as low as 140 nM and 60 nM for Cu2+ and Ag+ ions, respectively. The color change induced by fluorescent intensity ratio variation could also beemployed for visual discrimination. TheAuNC-embedded FS (FS-Au) nanoprobe was successfully used for Cu2+ and Ag+ ion determination in drinking water and intracellular Cu2+ imaging, which exhibits promising prospects in cost-effective and rapid determination of both Cu2+ and Ag+ with good sensitivity and selectivity.

A novel Aptamer-induced CHA amplification strategy for ultrasensitive detection of Staphylococcus aureus and NIR-triggered photothermal bactericidal Activity based on aptamer-modified magnetic Fe3O4@AuNRs

The development of multifunctional biosensors integrating recognition and therapeutic functions is critical for ensuring food safety and preventing foodborne diseases. Herein, an integrated design strategy was demonstrated to establish an innovative Fe3O4 @AuNRs-aptasensor for efficient detecting Staphylococcus aureus (S. aureus), which could enrich and photothermally eliminate bacteria under the radiation of near-infrared light irradiation (NIR). The detection assay relies on the binding of aptamer to Fe3O4 @AuNRs (Fe3O4 @ AuNRs-aptasensor) to induce the release of c-aptamer (c-Apt) in the presence of S. aureus, thereby triggering catalytic hairpin assembly (CHA) reaction and restoring the fluorescence of the reporter. The developed biosensor achieved direct, ultrasensitive, and specificity detection of S. aureus with a linear response ranging from 102 to 106 CFU/mL and a detection limit of 101 CFU/mL under optimal conditions. Furthermore, the aptasensor can be applied for the detection of S. aureus in milk with recoveries of 90.3–95.6% at the concentration between 1.0 × 102 and 1.0 × 104 CFU/mL. Additionally, Fe3O4 @AuNRs-aptasensor shows high photothermal sterilization performance under NIR irradiation. The study emphasizes the combination of high sensitivity base on molecular detection and photothermal antibacterial activity of novel nanomaterials activated by NIR light to design multifunctional probe, which show great potential for application in food analysis and early foodborne disease diagnosis.

Molecular Engineering of Near-Infrared Fluorescent Probes for Cell Membrane Imaging.

Cell membrane (CM) is a phospholipid bilayer that maintains integrity of a whole cell and relates to many physiological and pathological processes. Developing CM imaging tools is a feasible method for visualizing membrane-related events. In recent decades, small-molecular fluorescent probes in the near-infrared (NIR) region have been pursued extensively for CM staining to investigate its functions and related events. In this review, we summarize development of such probes from the aspect of design principles, CM-targeting mechanisms and biological applications. Moreover, at the end of this review, the challenges and future research directions in designing NIR CM-targeting probes are discussed. This review indicates that more efforts are required to design activatable NIR CM-targeting probes, easily prepared and biocompatible probes with long retention time regarding CM, super-resolution imaging probes for monitoring CM nanoscale organization and multifunctional probes with imaging and phototherapy effects.

Phenothiazine-based multifunctional fluorescent probe for one-site rapid detection of hydrazine in cells, soil, water samples and test strip

Hydrazine is widely used as a wide range of industrial chemical reagents. Because of its toxicity and explosiveness, it poses a serious threat to human health and ecological security. Therefore, it is of great significance to develop a real-time tracing method for hydrazine. Here, a novel turn-on fluorescent probe SXNU-1 for the detection of hydrazine was synthesized based on phenothiazine. SXNU-1 showed excellent selectivity toward hydrazine over various competitive species. Vinyl group with cyano and ethylacetate groups in SXNU-1 could react with hydrazine to form Schiff's base, showing bright green fluorescence. The response mechanism was verified by theoretical calculation, 1H NMR titration and HRMS. In addition, SXNU-1 can determine hydrazine in soil, water samples and test strip. Because SXNU-1 has good cell membrane permeability, it can monitor different concentrations of hydrazine in HeLa cells. Therefore, we believe that SXNU-1 has great potential in the analysis and monitoring of environmental pollutants.

Aggregation-induced bioprobe for plasma membrane-specific imaging and photodynamic cancer cell ablation

Selective labelling of the plasma membrane (PM) by fluorescence imaging techniques enables an intuitive analysis of cell status together with dynamic changes, and therefore is of great value. We herein disclose a novel carbazole-based probe, CPPPy, that shows aggregation-induced emission (AIE) property and is observed to selectively accumulate at the PM of living cells. Benefiting from its good biocompatibility and PM-targeted specificity, CPPPy can light up the PM of cells by high-resolution imaging even at a low concentration of 200 nM. Simultaneously, CPPPy is capable of generating both singlet oxygen and free radical-dominated species upon visible light irradiation, which further induces irreversible growth inhibition and necrocytosis of tumor cells. This study thus provides new insight into the construction of multifunctional fluorescence probes with PM-specific bioimaging and photodynamic therapy.

Ultra-sensitive fluorescent and colorimetric probes for femtomolar detection of picric acid: Mechanochromic, latent fingerprinting, and pH responsive character with AIE properties

• Probes 1(a-c) were synthesized through one-pot multicomponent reaction. • This study offers design strategy to transform ACQ into AIE feature. • Probes showed mechanochromism, pH responsive, and identification of latent fingerprints. • Probes displayed ultrasensitive femtomolar level sensing of picric acid (PA) • Selective sensing of PA was achieved through the predominant involvement of ground state complexation. Multiple stimuli responsive easy to synthesize organic probes with remarkable structural properties are imminently essential. To this endeavor, 1,4-dihydropyridine (DHP) based three new probes 1(a-c) were successfully synthesized and characterized through modern spectroscopic and mass spectrometry techniques. Planar dihydropyridine was appended with multiple donor-acceptor and rotors to bring aggregation induced emission (AIE) character in probes 1(a-b) . Interestingly, probe 1c displayed aggregation caused quenching (ACQ) effect due to perpendicularly attached naphthyl ring. Therefore, this study presents extensive structure-property relationship to invoke AIE character. Moreover, probes 1(a-c) exhibited high photostability, reversible mechanochromism, pH responsive character, and adequate identification of latent fingerprints (LFPs). Importantly, multifunctional probes 1(a-c) served as highly selective fluorescent and colorimetric probes for picric acid (PA). In solution, highly sensitive femtomolar (fM) level (limit of detection (LOD); 12.3, 14.6, and 27.1 fM) detection of PA was attributed to combined effect of ground-state charge transfer complex formation and Forster resonance energy transfer (FRET). Favorable complexation between probes and PA was proved through UV-visible absorption, density functional theory (DFT), and dynamic light scattering (DLS) studies. Advantageously, probes were able to detect picric acid in vapor phase. In addition, probes were practically applicable for the contact mode detection of PA and sensing of PA in LFPs and industrial samples.

Antibody Functionalization of Ultrasmall Fluorescent Core–Shell Aluminosilicate Nanoparticle Probes for Advanced Intracellular Labeling and Optical Super Resolution Microscopy

Fluorescent labeling of cellular substructures is commonly performed using antibody–organic dye conjugates. Organic dyes do not exhibit ideal optical properties in terms of brightness and photostability, however, in particular when it comes to advanced optical super-resolution microscopy (SRM) applications. Here, we demonstrate the efficient conjugation of widely available secondary antibodies and cationic species to ultrasmall (sub-10 nm) fluorescent silica core─poly(ethylene glycol) shell (core–shell) aluminosilicate nanoparticles (aC′ dots) encapsulating different color dyes for specific targeting and high-quality fluorescence imaging of structures of the cytoskeleton (tubulin and actin) and nucleus, respectively. We show that the different color aC′ dots provide enhanced brightness and photostability relative to their parent dyes. As recently discovered, we further demonstrate that they exhibit photo-induced blinking with low ON–OFF duty cycles enabling optical SRM, for example, in the form of stochastic optical reconstruction microscopy (STORM), without the need for complex imaging setups or cocktails. After carefully optimizing Ab–aC′ dot conjugation as well as cell structure labeling protocols in fixed and permeabilized HeLa and MDA-MB-231 cells, we demonstrate three-color STORM and exemplify improved resolution compared to standard antibody–dye conjugates. This work paves the way to next-generation multifunctional optical probes based on ultrasmall silica nanoparticle platforms for advanced applications in bioimaging, nanomedicine, and beyond.

Multifunctional Cd-CP for fluorescence sensing of Cr(VI), MnO4−, acetylacetone and ascorbic acid in aqueous solutions

The development of multifunctional fluorescent chemosensors for the detection of multiple targets remains challenging but of great importance. In this paper, one novel coordination polymer (CP), denoted as [Cd2(edda)(phen)2]∙H2O (compound 1, H4edda = 5,5′ (ethane-1,2-diylbis(oxy)) diisophthalic acid, phen = 1,10-phenanthroline) is successfully designed and prepared under hydrothermal conditions. Structural analysis indicates that compound 1 possesses a one-dimensional (1D) double chain structure, then self-assembles into a three-dimensional (3D) supramolecular framework via π…π interactions between phen molecules. Interestingly, compound 1 is found to be tolerant in wide range of acidic to alkaline aqueous solutions (pH = 2–13). Fluorescent spectral investigations reveal that compound 1 exhibits highly selective and sensitive fluorescence responses toward MnO4−, Cr(VI) ions, acetylacetone (acac) and ascorbic acid (AA) by fluorescence quenching in the aqueous phase. The detection limits are in the very low range, reaching μM level for the detection of MnO4−, Cr(VI) ions, nM for AA and ppm for acac detection. The distinguished multi-responsive performance suggests compound 1 to be a potential multifunctional probe. Furthermore, the possible quenching mechanisms have also been systematically investigated in this work.

N-doped carbon dots as the multifunctional fluorescent probe for mercury ion, glutathione and pH detection

Recently, carbon dots (CDs) have exhibited promising applications in the fluorescence detection of various ions and biomolecules. In this work, one kind of nitrogen-doped CDs (N-CDs) with high fluorescence intensity was synthesized, characterized by transmission electron microscopy, x-ray diffraction, x-ray photoelectron spectroscopy, Fourier-transform infrared, UV–vis absorption spectra, and fluorescence spectra. The results show that the spherical and uniform N-CDs (quantum yield: 60.2%) have remarkable fluorescence properties and photostability, which makes N-CDs can be utilized as an ‘on-off-on’ sensor for Hg2+ and glutathione (GSH). In addition, the pH-sensitive behavior of N-CDs makes it also applicable to H+ detection under acid conditions (pKa = 3.53). The linear range of the ‘turn-off’ sensor detecting Hg2+ was 0.014–50 μM, with a 0.014 μM limit of detection (LOD). GSH was detected by the fluorescence ‘turn-on’ method with a linear range of 0.125–60 μM and a LOD of 0.125 μM. The outstanding performance of N-CDs makes it potential applications in ecological pollution and biomolecule visualization monitoring.

A novel lipid droplets/lysosomes-targeting colorimetric and ratiometric fluorescent probe for Cu2+ and its application

A novel multifunctional fluorescent probe LL2 was prepared via a one-step condensation reaction between 3-formyl-N-butylcarbazole and 2-Hydroxy-1-naphthylhydrazone. LL2 can work as a colorimetric probe for Cu2+, and can also selectively recognize Cu2+ via ratiometric fluorescence signal. After the addition of Cu2+, the probe LL2 responded rapidly within 5 s and reached stability within 30 s. In natural light, when Cu2+ were added to the solution, the color of probe LL2 changed from yellowish to colorless, while there was a discernible fluorescence changed from green to blue under a 365 nm UV lamp. The ratiometric fluorescence intensity (F449/F510) showed a good linear relationship (R2 = 0.9902) with Cu2+ concentration in the concentration range of 0–5 μmol/L, and the minimum detection limit was 1.96 μM. Cell imaging experiments showed that LL2 could capture fluorescence signals in the green and blue channels of HepG2 cells through fluorescence confocal microscope, and successfully recognize exogenous Cu2+ in HepG2 cells. In addition, fluorescence co-localization experiments showed that LL2 could target both lipid droplets and lysosomes. Meanwhile, LL2 could be applied to filter paper strip assay and detection of Cu2+ in actual water samples. These results indicated that probe LL2 has a good capability for monitoring Cu2+ in environment and living cells.

Laboratory experiments based on tip probe － Scanning probe detection and regulation of ferroelectric domains and their microscopic physical properties

<sec>Ferroelectric and multiferroic materials have gained significant attention due to their potential applications in investigating emergent cross-coupling phenomena among spin, charge, orbit, and lattice in correlated electron systems, as well as promising candidates for prospective applications in advanced industries, <i>e.g.</i> data memory/processing, sensors, actuators, and energy-relevant devices. The structure and dynamic characteristics of ferroelectric domains can significantly affect the physical properties and device functions of materials, such as electrical conductivity, photovoltaics, and magnetoelectric coupling, particularly, novel topological domains can bring many new physical properties. These make it possible to design materials and devices through domain engineering methods. Therefore, exploring the microdomain structures and related physical property is expected to bring new material and device solutions for post-Moore's era information technology.</sec><sec>Accurate understanding of domain structures and their corresponding functionalities pose challenges to characterization techniques. In particular, it remains challenging to investigate the dynamics and cross-coupling behaviors on a nanoscale <i>in situ</i>. Nowadays, it is worthwhile to pay more attention to the multifunctional scanning probe microscopy technique, as it serves as a versatile and powerful nanoscale probe capable of exploring multifunctionalities. Multi-field stimulation such as electric field, magnetic field, light illumination, strain field, and thermal field can be combined with the advanced scanning probe microscopy technique, making it an ideal platform for in-situ manipulation of domain structure and its related functional response on a nano-scale.</sec><sec>In this study, we give a brief overview on the recent advances in our research group in detection and manipulation of ferroelectric domains and microscopic physical properties through multifunctional scanning probe microscopy technique. Special attention is paid to those topological domain structures such as vortex, center domain state and bubble domain in size-confined systems (ultrathin films/multilayers and nanodots/nanoislands) and their associated novel physical phenomena. In addition, the controllability of electric field driven magnetic switching in multiferroic heterostructures is also studied through size effect, interfacial coupling and domain engineering. Finally, we present some suggestions for future directions. Most of these studies are conducted by using the tip probe, so it is named the “Laboratory experiments based on tip probe”.</sec>

Multifunctional activity-based chemical probes for sirtuins.

The sirtuin family of NAD+-dependent protein deacylases has gained significant attention during the last two decades, owing to their unique enzymatic activities as well as their critical roles in a broad array of cellular events. Innovative chemical probes are heavily pursued for the functional annotation and pharmacological perturbation of this group of "eraser" enzymes. We have developed several series of activity-based chemical probes (ABPs) to interrogate the functional state of active sirtuins in complex biological samples. They feature a simple Ala-Ala-Lys tripeptide backbone with a thioacyl "warhead", a photoaffinity group (benzophenone or diazirine), and a bioorthogonal group (terminal alkyne or azido) for conjugation to reporters. When applied in a comparative fashion, these probes reveal the changes of active sirtuin contents under different physiological conditions. Additionally, they can also be utilized in a competitive manner for inhibitor discovery. The Nobel-winning "click" conjugation to a fluorophore allows the visualization of the active enzymes, while the covalent adduct to a biotin leads to the affinity capture of the protein of interest. Furthermore, the "clickable" tag enables the easy access to proteolysis targeting chimeras (PROTACs) that effectively degrade human SIRT2 in HEK293 cells, albeit at micromolar concentrations. These small molecule probes offer unprecedented opportunities to investigate the biological functions and physiological relevance of the sirtuin family.

Multifunctional DNA nanoprobe for tumor-targeted synergistic therapy by integrating chemodynamic therapy with gene silencing.

Due to the high complexity, diversity and heterogeneity of tumor occurrence and development, multi-mode synergistic therapy is more effective than single treatment modes to improve the antitumor efficacy. Also, multifunctional probes are crucial to realize synergistic therapy. Herein, a multifunctional DNA tetrahedron nanoprobe was ingeniously designed to simultaneously achieve chemodynamic therapy (CDT) and gene silencing for synergistic antitumor. The multifunctional DNA tetrahedron nanoprobe, DNA tetrahedron-silver nanocluster-antagomir-21 (D-sgc8-DTNS-AgNCs-Anta-21), integrated a CDT reagent (DNA-AgNCs) and miRNA-21 inhibitor (Anta-21) with a specific recognition probe (aptamer). After targeted entry in cancer cells, D-sgc8-DTNS-AgNCs-Anta-21 silenced endogenous miRNA-21 by Anta-21 and produced highly toxic ˙OH by reacting with H2O2, which induced apoptosis in the tumor cells. The targeted recognition of aptamers led to the concentration-dependent death of HeLa cells. On the contrary, the cell survival rate of normal cells was basically unaffected with an increase in the concentration of D-sgc8-DTNS-AgNCs-Anta-21. Therefore, the diverse functions, biocompatibility and programmability of DNA provide a useful and easy way to assemble multifunctional probes for synergistic therapy.

Synthesis of a novel triphenylamine-based multifunctional fluorescent probe for continuous recognition applications

In this paper, a novel fluorescent probe, TPA-PAT, with continuous recognition based on triphenylamine was designed, synthesized, and characterized by NMR, IR, and fluorescence spectrophotometry techniques.

One step synthesis of highly photoluminescent red light-emitting carbon dots from O-phenylenediamine and 2,4-diaminophenol as fluorescent probes for the detection of pH and Cr(VI).

In this study, new red light-emitting carbon dots (R-CDs) that can selectively recognize Cr(VI) were prepared using a strategy that utilizes 2,4-diaminophenol to enhance the fluorescence of O-phenylenediamine based carbon dots. The results showed that 2,4-diaminophenol increased the quantum yield (QY) of the carbon quantum dots (CDs), and that the QY of the CDs increased from their original value of 8.7% to 20.1% (R-CDs). The R-CDs show sensitivity to acidic conditions and maintain good linearity between pH = 1.00-4.00, making them useful as pH probes. Furthermore, the prepared R-CDs possess good solubility in water and are responsive to changes in Cr(VI) concentrations in aqueous environments. The quenching of the R-CDs fluorescence was linearly correlated with the Cr(VI) concentration within a range of 0-20 μM, with a lower detection limit of 66 nM. The detection mechanism is attributed to the formation of hydrogen bonds between Cr(VI) and the R-CDs, resulting in the fluorescence quenching of the R-CDs. The R-CDs can be considered effective multifunctional fluorescent probes for both pH and Cr(VI) in aqueous environments. This study will provide new R-CD design strategies for probes that selectively identify specific target substances.

Unique development of a new dual application probe for selective detection of antiparallel G-quadruplex sequences.

G-Quadruplex (G4) structures play vital roles in many biological processes; consequently, they have been implicated in various human diseases like cancer, Alzheimer's disease etc. The selective detection of G4 DNA structures is of great interest for understanding their roles and biological functions. Hence, development of multifunctional fluorescent probes is indeed essential. In this investigation, we have synthesized a quinolinium based dual application probe (QnMF) that presents molecular rotor properties. This dual application molecular rotor is able to detect selectively antiparallel G4 sequences (22AG in 100 mM NaCl) through a turn-on response over other G4 topologies. The QnMF also contains a distinct fluorine-19 that undergoes a significant chemical shift in response to microenvironmental changes around the molecule when bound to G4 structures. The probe QnMF exhibits significantly brighter fluorescence emissions in glycerol (ε × ϕ = 2800 cm-1 M-1) and relatively less brighter fluorescence emissions in methanol (ε × ϕ = 40.5 cm-1 M-1). The restricted rotation inherent property of the QnMF molecular rotor is responsible for brighter fluorescence and leads to enhancement in the fluorescence upon binding to the G4 structure. Overall, the probe's dual detection method makes it useful for monitoring the G4 structures that are abundant and plays a vital role in living organisms.

An off-on-off fluorescence probe based on a pyrazole derivative for Al3+ and Fe3+ detection

A multifunctional fluorescence probe (L) was synthesized and used as a dual sensing platform for detecting Al3+ and Fe3+.