Light-up Probe Research Articles

A Radical-Generating Probe to Release Free Fluorophores and Identify Artemisinin-Sensitive Cancer Cells.

The smart light-up probes have been extensively developed to image various enzymes and other bioactive molecules. Upon activation, these probes result in light-up fluorophores that exist in a protein-bound or a free form. The difference between these two forms has not yet been reported. Here, we present a pair of smart light-up probes that generate a protein-bound fluorophore and a free fluorophore upon activation by heme. Probe 8 generated a radical-attached fluorophore that predominantly existed in the free form, while probe 10 generated an α,β-unsaturated ketone-attached fluorophore that showed extensive labeling of proteins. In live-cell imaging, probe 8 showed greater fluorescence intensity than probe 10 when low concentrations (0.1-5 μM) of the probes were used, but probe 8 was less fluorescent than probe 10 when the concentrations of the probes were high (10 μM). Finally, probe 8 was used to reflect the activation level of the endoperoxide bond in cancer cells and to effectively distinguish ART-sensitive cancer cells from ART-insensitive ones.

Understanding the role of π-π stacking from red carbon for selective, high stable and anti-external interference detecting ferric ions and cellular imaging

The subcarboxylated carbon (C3O2) is a unique molecule that spontaneously polymerizes into highly conjugated fluorescence structures p(C3O2)n, named “red carbon”. This unique property, combined with its role as aggregation-induced emission luminogens, has allowed for a deeper exploration of their emission mechanism. Herein, both density functional theory calculations and experimental approaches to elucidate the crystal structure of π–π stacking in red carbon, thereby enhancing the fluorescence properties of p(C3O2)n polymer in its aggregated state. The time-dependent density functional theory calculations and experimental observations indicate that the fluorescence of red carbon can be quenched by ferric ions (Fe3+) and subsequently restored by ethylenediaminetetraaceticacid (EDTA). This quenching process follows first-order reaction kinetics. On the basis of the coordination to metal ions from abundant heteroatoms on red carbon, the high sensitivity, quick responsive (1 min), sensitive detection (the lowest detection limit is 0.97 μM) for the Fe3+ were realized in red carbon, which provides inspirations for the design of new turn-on probes with high stability in detecting the Fe3+ in aqueous solution. Crucially, the red carbon as a light-up fluorescence probe can be employed for the Fe3+ sensing in living cells, which enlighten a new perspective for designing non-conjugated fluorescent polymers. This innovative approach opens up new avenues for developing probes that can detect Fe3+ with high precision and reliability in complex biological environments.

Design of synthetic peptide-based fluorescence probes for turn-on detection of hyaluronan

Herein, we designed and examined a series of fluorescent peptide-based probes for turn-on detection of hyaluronan (HA), a member of the glycosaminoglycan family. We utilized two kinds of synthetic HA-binding peptides as the binding unit for HA, and each peptide was coupled with three kinds of environment-sensitive fluorophores as the signaling unit. From the examination of the peptides, fluorophores, and the position and number of fluorophore modification, we found that X7 peptide (RYPISRPRKR) labelled with an aggregation-induced emission (AIE) fluorogen, tetraphenylethene (TPE), at the N-terminal (named TPE-X7) did function as a light-up probe for HA. The response of TPE-X7 was highly selective to higher molecular weight HA in comparison with lower ones, having the possible potential for the analysis of HA size. TPE-X7 was also applicable to the quantification of HA in synovial fluids.Graphical abstract

G-quadruplex in mitochondria as a possible biomarker for mitophagy detection

Mitochondrial autophagy (mitophagy) is a key physiological process that maintains the homeostasis of mitochondrial quality and quantity. Monitoring mitophagy is of great significance for detecting cellular abnormalities and developing therapeutic drugs. However, there are still very few biomarkers specifically developed for monitoring mitophagy. Here, we propose for the first time that mitochondrial G-quadruplex may serve as a biomarker for mitophagy detection, and develope a fluorescent light-up probe AMTC to monitor mitophagy in live cells. During mitophagy, AMTC fluorescence is significantly enhanced, but once mitophagy is inhibited, its fluorescence immediately decreases. The fluorescence behavior of AMTC implicates an increase in the formation of mitochondrial G-quadruplex during mitophagy. This inference has also been supported by the other two G-quadruplex probes. Taken together, this work provides a new possible biomarker and detection tool for the study of mitophagy.

Nucleic acid detection with single-base specificity integrating isothermal amplification and light-up aptamer probes.

We report a novel platform for label-free nucleic acid detection using isothermal amplification and light-up aptamer probes. This assay converts double-stranded amplicons into single-stranded targets to enable sequence-specific hybridization with split dapoxyl aptamer probes, offering attomolar sensitivity and single-base specificity.

In-gel staining methods of G4 DNA and RNA structures.

G-quadruplexes (G4) are functionally important nucleic acid structures, involved in many cellular pathways. They are often dynamically regulated in cells, which makes detecting them in vivo challenging and dependent on sophisticated technical equipment. Therefore, in vitro studies are commonly performed as a first step to confirm a candidate sequence folds into a G4. Several methods have been developed, each with its individual pros and cons. A highly accessible and quick approach, without the need for specialized equipment, is the detection of G4s in native gels using light-up probes. These molecules become fluorescent after specifically binding to G4s. Several different classes have been discovered, emitting light in various colors, and some possess specificity for certain G4 topologies, which makes them highly versatile tools for G4 visualization. Here, we will explore the general procedure using the light-up probe NMM on RNA G4s and discuss advantages and limitations of this method.

Design of a sustainable light-up flavonol probe for dual-ratiometric fluorescent sensing and visual differentiating ammonia and hydrazine.

Ammonia (NH3) and hydrazine (N2H4) present potential risks to human health, food and environmental safety. A sustainable flavonol-based probe, quercetin pentaacetate (QPA, weak blue emission 417nm), was fabricated for dual-ratiometric fluorescent sensing and visual differentiating NH3 and N2H4. Excited state intramolecular proton transfer-on products with green (487nm) and yellow (543nm) emissions occurred as meeting with NH3 and N2H4, respectively, for their different nucleophilicities. Such a promising response offered a great opportunity of QPA to discriminatively detect NH3 and N2H4 with large Stokes shifts (>122nm), high sensitivity (limit of detection: 35.4μM and 0.70ppm for NH3 solution and gas; 0.26μM for N2H4 solution), excellent accuracy (spiked recoveries from 98.6 % to 105 %), and superior selectivity. Importantly, QPA was utilized for monitoring NH3 vapor in fish spoilage procedures and detecting N2H4 in water samples for food and environmental safety evaluation.

Lighting up trace carbon monoxide and residual palladium species by a low cytotoxic mitochondria targetable red fluorescent probe: Its large scaled applications.

High levels of residual palladium can lead to serious negative health effects. Carbon monoxide (CO) is a significant gasotransmitter in transporting intermolecular and intramolecular signals to balance several physiological processes. Therefore, there is a need for rapid detection of CO and palladium residue. To address these issues, we have designed a novel light-up fluorescent probe for the detection of Pd and CO. It can not only detect Pd and CO selectively with a remarkable chromogenic and red fluorescent response over other metal ions allowing detection with naked eyes but also discriminate Pd0 and Pd2+/Pd4+ species. The detection reaction is confirmed by HPLC analysis. The probe demonstrates biocompatibility and mitochondrial target ability for potential biological applications. The practical applications based on drug residue and soil analysis, and smartphone have been successfully performed. Bioimaging of the concentration change of Pd and CO in HeLa cells using the probe is successfully applied. Therefore, the present approach can provide early diagnosis of Pd and CO with low detection limit, low cytotoxicity, high selectivity, and sensitivity.

A light-up fluorescence probe for wash-free analysis of Mu-opioid receptor and ligand-binding events

With the aggravated burden of opioid use disorder spreading worldwide, demands for new forms of opioid receptor agonist/antagonist constitute immense research interest. The Mu-opioid receptor (MOR) is currently in the spotlight on account of its general involvement in opioid-induced antinociception, tolerance and dependence. MOR binding assay, however, is often complicated by difficulty in MOR separation and purification, as well as the tedious procedure in standard biolayer interferometry and surface plasmon resonance measurements. To this end, we present TPE2N as a light-up fluorescent probe for MOR, which exhibits satisfactory performance in both live cells and lysates. TPE2N was elaborately designed based on the synergistic effect of twisted intramolecular charge-transfer and aggregation-induced emission by incorporating a tetraphenylethene unit to emit strong fluorescence in a restrained environment upon binding with MOR through the naloxone pharmacore. The developed assay enabled high-throughput screening of a compound library, and successfully identified three ligands as lead compounds for further development.

Development of a novel light-up probe for detection of G-quadruplexes in stress granules

G-quadruplexes (G4s) regulate various biological processes in cells. However, cellular imaging of dynamically forming G4s in biomolecular condensates using small molecules has been poorly investigated. Herein, we present a fluorescent light-up probe with the ability to selectively stabilize G4s and enhance fluorescence upon G4 binding. The foci of the probe were mainly observed in the nucleoli. These were co-localized with anti-fibrillarin antibodies and anti-G4 antibodies (BG4). Moreover, we tested detection of G4 in stress granules using the developed probe. Stress granules were induced through treatment with not only thapsigargin, but also known G4 ligands (pyridostatin, RHPS4, and BRACO-19). In the stress granules, co-localization between the probe, BG4, and stress granule markers (TIA1 and G3BP1) was detected. We present a practical light-up probe for G4s in stress granules, providing potential targets for G4 ligands.

Theoretical investigation on the fluorescent sensing mechanism for recognizing formaldehyde: TDDFT calculation and excited-state nonadiabatic dynamics

Inspired by the activity-based sensing method, the hydrazine-modified naphthalene derivative (Naph1) was synthesized and used as a fluorescent probe to detect formaldehyde (FA) in living cells. Through the condensation reaction between the probe Naph1 and analyte FA, researchers observed a ∼14 folds enhancement of fluorescent signal around 510 nm in an experiment, realizing the high selectivity and sensitivity detection of FA. However, a theoretical understanding of the sensing mechanism was not provided in the experimental work. Given this, the light-up fluorescent detecting mechanism was in-depth unveiled by performing the time-dependent density functional theory (TDDFT) and the complete active space self-consistent field (CASSCF) theoretical calculations on excited-state intramolecular proton transfer (ESIPT) and non-adiabatic excited-state dynamics simulation. The deactivation channel of S1/T2 intersystem crossing (ISC) was turned off to successfully recognize FA. Insight into the ESIPT-based fluorescent detecting mechanism indicated that ESIPT was essential to light-up fluorescent probes. This work would provide a new viewpoint to develop ESIPT-based fluorescent probes for detecting reactive carbon species in vivo or vitio.

Bright and Light-Up Sensing of Benzo[c,d]indole-oxazolopyridine Cyanine Dye for RNA and Its Application to Highly Sensitive Imaging of Nucleolar RNA in Living Cells.

Small molecular weight probes that can show a fluorescence signaling response upon binding to RNAs are promising for RNA imaging in living cells. Live-cell RNA imaging probes that can achieve a large light-up ability (>100-fold) and high Φbound value for RNA (>0.50) have been rarely reported to date. Here, benzo[c,d]indole-oxazolopyridine (BIOP), an unsymmetrical monomethine cyanine analogue, was newly developed as a bright and large light-up probe for imaging of nucleolar RNA in living cells. BIOP served as a yellow-emissive probe (λem = 570 nm) and exhibited a significant light-up response upon RNA binding (770-fold) with a high Φbound value (0.52). We demonstrated the advantages of BIOP over a commercially available RNA-staining probe, SYTO RNA select, for robust and sensitive RNA sensing by a systematic comparison of fluorescent properties for RNA. In addition, BIOP was found to possess high membrane permeability and low cytotoxicity in living cells. The examination of live-cell imaging revealed that BIOP exhibited emission in the nucleolus upon binding to nucleolar RNA much stronger than that of SYTO RNA select. Furthermore, BIOP facilitated the highly sensitive imaging of nucleolar RNA, in which 50 nM BIOP can stain nucleolar RNA in living cells with a 20 min incubation.

Bioorthogonal Light-Up Fluorescent Probe Enables Wash-Free Real-Time Dynamic Monitoring of Cellular Glucose Uptake.

As a significant energy source for living systems, the aberrant cellular glucose uptake is seriously implicated in numerous metabolic diseases. Unfortunately, current shortage of robust tools leaves the limitation to understand its precise biology. Herein we presented a bioorthogonal light-up fluorescent probe consist of two reagents, Glu-HT-Me+AzGlu2, for rapidly responsive (within 25 min), highly specific and sensitive (20-folds enhancement) detection of live-cell glucose uptake based on arylphosphine-induced a-PET effect and Staudinger ligation. Especially, taking the advantage of wash-free characteristic, the probe displayed the real-time dynamic monitoring of cellular glucose uptake. Furthermore, it was successfully capable of not only differentiating cancer cells from normal cells, but also allowing evaluation of anticancer/glycolysis/transport mediated glucose flux. Importantly, it was employed to monitor the fluctuations of glucose uptake in a doxycycline-inducible K-rasG12V expression oncogenic cell system, implying its potential as a valuable tool to explore glucose uptake biology.

A novel TICT-based near-infrared fluorescent probe for light-up sensing and imaging of human serum albumin in real samples

Human serum albumin (HSA) has emerged as a pivotal biomarker and prognostic indicator for various human diseases. Real-time sensing and visual tracking of HSA in plasma or other biological systems will immensely facilitate the basic researchers and clinicians to better understand HSA-associated biological processes. Herein, a novel near-infrared (NIR) fluorescent probe (7-HTCF) was rationally constructed for light-up sensing and in-situ imaging of HSA in real samples, based on the principle of twisted intramolecular charge transfer (TICT). Under physiological conditions, 7-HTCF could be efficiently trapped by HSA to form a stable complex via binding on a non-drug binding site, while the complex emitted strong fluoresce signals around 670 nm. Further investigations demonstrated that 7-HTCF displayed a great combination of excellent selectivity and good chemical stability, as well as rapid fluorescent response and ultra-high sensitivity for HSA detection. Particularly, the newly developed light-up probe has been successfully utilized for quantitative detection of HSA in diluted plasma samples, while its readouts are hardly affected by the addition of therapeutic agents and herbal medicines. 7-HTCF is also successfully used for in-situ imaging of the reabsorbed HSA in living renal cells, while this dye exhibits good cell permeability and high resolution for in-situ imaging in living cells. Collectively, a novel TICT-based near-infrared fluorescent probe was devised for highly selective and ultra-sensitive sensing of HSA in plasma samples or imaging HSA in living cells, which offered a practical tool for clinical tests and for exploring HSA-associated biological processes.

A highly selective and sensitive fluorescent light-up probe for rapid detection of mercury ions in aqueous solution

Due to its complex biological toxicity, mercury ions can cause various neurological diseases and even death. Therefore, developing a rapid and efficient mercury ions analysis method is of great significance. In this study, we designed a new water-soluble fluorescent chemical sensor, Hg535, which can sensitively detect mercury ions in an aqueous solution. Based on the specific mercury-mediated desulfurization reaction, the probe exhibits high selectivity for mercury ions, excellent sensitivity, significant Stokes shift (108 nm), and fast kinetics (within 15 s), with a low detection limit (1.66 ppb). Finally, agar gels based on the probe Hg535 were fabricated, which could act as a solid optical sensor to conveniently and efficiently detect Hg2+.

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.

Ruthenium(II) Polypyridyl Complexes and Their Use as Probes and Photoreactive Agents for G-quadruplexes Labelling.

Due to their optical and electrochemical properties, ruthenium(II) polypyridyl complexes have been used in a wide array of applications. Since the discovery of the light-switch ON effect of [Ru(bpy)2dppz]2+ when interacting with DNA, the design of new Ru(II) complexes as light-up probes for specific regions of DNA has been intensively explored. Amongst them, G-quadruplexes (G4s) are of particular interest. These structures formed by guanine-rich parts of DNA and RNA may be associated with a wide range of biological events. However, locating them and understanding their implications in biological pathways has proven challenging. Elegant approaches to tackle this challenge relies on the use of photoprobes capable of marking, reversibly or irreversibly, these G4s. Indeed, Ru(II) complexes containing ancillary π-deficient TAP ligands can create a covalently linked adduct with G4s after a photoinduced electron transfer from a guanine residue to the excited complex. Through careful design of the ligands, high selectivity of interaction with G4 structures can be achieved. This allows the creation of specific Ru(II) light-up probes and photoreactive agents for G4 labelling, which is at the core of this review composed of an introduction dedicated to a brief description of G-quadruplex structures and two main sections. The first one will provide a general picture of ligands and metal complexes interacting with G4s. The second one will focus on an exhaustive and comprehensive overview of the interactions and (photo)reactions of Ru(II) complexes with G4s.

One-step light-up metabolic probes for in situ discrimination and killing of intracellular bacteria

TPEPy-Ala and TPAPy-Kdo with metabolic moieties can be directly incorporated into the bacterial cell envelopes and light up intracellular bacteria. Additionally, the metabolic probes can effectively eliminate labeled bacteria in situ with minimal host cell cytotoxicity via photodyanmic therapy.

Tetraphenylethene derivative that discriminates parallel G-quadruplexes.

G-Quadruplex (G4), as a non-canonical nucleic acid secondary structure, has been proved to be prevalent in genomes and plays important roles in many biological processes. Ligands targeting G4, especially small-molecular fluorescent light-up probes with selectivity for special conformations, are essential for studying the relationship between G4 folding and the cellular response. However, their development still remains challenging but is attracting massive attention. Here, we synthesized a new tetraphenylethene derivative, namely TPE-B, as a parallel G4 probe. Fluorescence experiments showed that TPE-B could give out a strong fluorescence response to the G4 structure. Moreover, it gave a much higher fluorescence intensity response to parallel G4s than anti-parallel ones, which indicated that TPE-B could serve as a special tool for probing parallel G4s. The circular dichroism (CD) spectra and melting curves showed that TPE-B could selectively bind and stabilize parallel G4s without changing their topology. ESI-MS studies showed that TPE-B could bind to parallel G4 with a 1 : 1 stoichiometry. The gel staining results showed that TPE-B was a good candidate for probing parallel G4s. Altogether, the TPE-B molecule may serve as a promising new probe that can discriminate parallel G4s.

Selective c-MYC G4 DNA recognition based on a fluorescent light-up probe with disaggregation-induced emission characteristics.

The c-MYC promoter is well-known as an important oncogene, the overexpression of which leads to ∼80% of all solid tumors. The four-stranded G4 present in the c-MYC promoter has been shown to play a pivotal role in the regulation of c-MYC transcription. Accordingly, strategies employed for c-MYC G4 DNA sensing have implications for the detection of many human pathologies. However, achieving specificity toward c-MYC G4 over other structurally similar G4s is a challenging task. Here, a supramolecular strategy that relies on the recognition-driven disaggregation of a novel BODIPY probe is outlined. The synthesized probe remained almost non-fluorescent in aqueous media in the aggregation state. Of all the tested G4 and non-G4 DNAs, only c-MYC triggered probe disaggregation and induced a significant increase in fluorescence intensity. The binding details discussed here suggest the basis for the recognition of a particular G4 structure, thus opening up a new way for the design and development of sequence-selective supramolecular G4 probes with desired properties.