Probe P1 Research Articles

A naphthimide based fluorescent probe for the detection of thiophenols and its application in actual water and food

Thiophenol (PhSH) and its derivatives are widely employed as common industrial raw materials, while they also pose high toxicity for animals and aquatic organisms. Herein, a novel fluorescent probe P1 was constructed, based on photo-induced electron transfer (PET) mechanism, using naphthimide as the fluorophore and 7-nitrobenzoxadiazole as the recognizing group. Probe P1 exhibited an “Off-On” fluorescence recognition response to PhSH by nucleophilic substitution reaction. The probe displayed advantageous properties for the recognition of PhSH, including a chromogenic response, high selectivity, fast response (< 90 s), and a large Stokes shift (150 nm). In addition, probe P1 exhibited a good linear response to PhSH within the range of 0–9 μM, and the limit of detection (LOD) is 21 nM. Furthermore, probe P1 has been successfully applied for the efficient and rapid PhSH detection in environmental water and actual food samples.

Mitochondria-targeted fluorescent probes based on coumarin-hemicyanine for viscosity changes and their applications in cells and mice

As an important parameter of the cellular microenvironment, the changes in mitochondrial viscosity are closely related to various life activities. Therefore, the development of fluorescent probes for test the changes of mitochondrial viscosity has great significance. In this study, we developed two fluorescent probes for the detection of the mitochondrial viscosity changes. The probes exhibited different fluorescence intensities at different viscosity based on the twisted intramolecular charge transfer process. The characteristics of high anti-interference performance, wide pH applicability, low cytotoxicity and excellent mitochondrial targeting performance made the probes successfully used to distinguish normal cells from cancer cells, achieving visualization of viscosity changes. Furthermore, probes P1 and P2 can also be used as early diagnosis of tumors in mice and reveal the pathology of tumor development. The probes could be serve as a promising viscosity detection tool for discriminating normal cells and cancer cells in biology-related research.

Dual Detection of microRNAs by a Signal-Off Colorimetric Nanobiosensor Based on Novel Split DNAzyme Nanostructure.

Breast cancer is the most common cancer and the second cause of cancer-related death in women, especially in the age of 20-59years. It is very important to diagnose it in the early stages of development due to high chance of survival. In this research, the early detection of two microRNAs involved in breast cancer including miR-21 and miR-155 was performed simultaneously using a nanobiosensor based on a special G-quadruplex structure and a colorimetric manner. This nanobiosensor contains two probes (p1, p2) that play a role in the formation of a special structure called DNA-G4. This structure has peroxidase-like properties and can oxidize TMB and produce a blue color. The diagnostic method is designed as a signal off, where the hybridization of probes with microRNA sequences, no DNA-G4 structure is formed and no color change is observed. The results of this study showed that the linear range of response is in the range of 2 to 10nm and limit of detection in buffer, blood and urine samples was calculated as 0.43nM, 0.54nM, and 0.62nM (R2 = 0.98), respectively. Evaluation using the method for cancer monitoring can be a simple, fast and cost-effective technique.

A dual responsive bis-thiophene affixed thiosemicarbazide based chemosensor for colorimetrically Hg2+ and fluorometrically Cu2+ ions and their applications in live cell imaging

In this work, we developed a fast and straightforward colorimetric and photoluminescent chemosensor probe (P1), featuring bis-thiophene-thiosemicarbazide moieties as its signaling and binding unit. This probe exhibited rapid sensitivity to Hg2+ and Cu2+ ions in a semi-aqueous medium, resulting in distinct colorimetric and photoluminescent changes. In the presence of Cu2+, P1 displayed an impressive 50-fold increase in photoluminescence (PL) at 450 nm (with excitation at 365 nm). The probe P1 formed a 1:1 complex with Hg2+ and Cu2+ ions, featuring association constant values of 4.04 × 104 M−1 and 1.25 × 103 M−1, respectively. P1 has demonstrated its efficacy in the analysis of real samples, yielding promising results. Additionally, the probe successfully visualized copper ions on a mouse fibroblast cell line (NIH3T3), highlighting its potential as an intracellular probe for copper ion detection.

Improved Affinity: A Customized Fluorescent Probe for the Rapid Detection of Butyrylcholinesterase

This article presents the distinctive butyrylcholinesterase (BChE) fluorescent probe P5, designed via a targeting-site method. This method was proposed to enhance the affinity of the probe for BChE by targeting the peripheral anionic site (PAS) of BChE. By mimicking the natural substrate butyrylcholine, the structure of the probe was optimized by introducing a positive charge. Fluorescent probe P5, selected from a series of designed fluorescent probes P1–P6, exhibited excellent affinity and specificity towards BChE, enabling rapid detection within 5 min with a low detection limit of 16.7 ng/mL. Furthermore, this probe can distinguish between normal cells and Alzheimer’s disease (AD) model cells, and demonstrated good imaging results in a P12 cell AD model. The results of this study indicate that this novel fluorescent probe could serve as a promising tool for the rapid detection of BChE and accurate AD diagnosis.

Polarity-Driven Fluorescence Monitoring of Lipid Droplet Dynamics in Dry Eye Disease.

The emergence of lipid droplets (LDs) has been recognized as cellular markers of ocular surface hyperosmosis, which is recognized as a fundamental mechanism driving dry eye disease (DED), while their dynamics during DED progression and therapy remains unlocked. For this purpose, an LD-specific fluorescent probe P1 is presented in this work that exhibits highly selective and sensitive emission enhancement in response to a decreased ambient polarity (Δf) from 0.209 to 0.021. The hydrophobic nature of P1 enables specific staining of LDs, facilitating visualization of changes in polarity within these cellular structures. Utilizing P1, we observe a decrease in polarity accompanied by an increase in the size and number of LDs in hyperosmotic human corneal epithelial cells (HCECs). Furthermore, interplays between LDs and cellular organelles such as mitochondria and the Golgi apparatus are visualized, suggesting the underlying pathogenesis in DED. Notably, the variations of LDs are observed after the inhibition of ferroptosis or activation of autophagy in hyperosmotic HCECs, implying the great potential of LDs as indicators for the design and efficacy evaluation of DED drugs regarding ferroptosis or autophagy as targets. Finally, LDs are confirmed to be overproduced in corneal tissues from DED mice, and the application of clinical eye drops effectively impedes these changes. This detailed exploration underscores the significant roles of LDs as an indicator for the deep insight into DED advancement and therapy.

Bifunctional fluorescent probe for fluoride and copper(Ⅱ) based on perylene diimide derivative and its application in cell imaging

A bifunctional fluorescent probe P1 based on perylene diimide derivative (PDI) was developed, demonstrating high sensitivity and selectivity for the rapid detection of fluoride (F-) and copper (Cu2+). The fluorescence of P1 was quenched upon interaction with F- and enhanced upon interaction with Cu2+. The concentration ranges for fluorescent detection of F- and Cu2+ are 0 ∼ 4 × 10-5 M and 0 ∼ 2.4 × 10-5 M, respectively. The stoichiometry of the complex formed between Cu2+ and P1 or F− and P1 was 1:1. The probe displays detection limits of 0.16 and 0.21 μM and binding constants (Ksv) of 3.92 × 104 and 1.72 × 104 M−1 for the detection of F- and Cu2+, respectively. The sensing mechanism and binding mode of P1 with F- and Cu2+ were investigated. The fluorescence quenching/enhancement of P1 was controlled by a photoinduced electron-transfer (PET) process. Furthermore, the ability of the probe to detect intracellular F- and Cu2+ was assessed using cell imaging techniques.

A novel coumarin-linked tetraphenylethene fluorescent probe for simultaneous sensing of ATP and GSH

Adenosine triphosphate (ATP) as the intracellular energy currency, and glutathione (GSH) as the regulator of redox homeostasis in the cell microenvironment, are all closely associated with numerous important physiological processes in living organisms. However, the fluorescent probes that can simultaneously detect ATP and GSH are rarely reported. Herein, a bifunctional coumarin-linked tetraphenylethene (TPE) probe P1 was successfully developed, which exhibits a red emission at 633 nm owing to the fluorescence resonance energy transfer (FRET) from energy donor coumarin to energy acceptor TPE. P1 can detect ATP with a fluorescence turn-on response at 633 nm in the red channel by complexation-enhanced aggregation mechanism, and showed a ratiometric detection toward GSH through the disruption of the FRET process, accompanied by an increase in the blue channel at 467 nm and a decrease in the red channel at 633 nm. P1 also displays a superior sensitivity and high selectivity to ATP and GSH, with a detection limit of 20.80 nM for ATP and 3.47 µM for GSH. Significantly, P1 can discriminate GSH from ATP and other analytes due to the different response times. Furthermore, P1 can localize mitochondria and has been discovered to be capable of imaging ATP and GSH in living cells and zebrafish.

Ratiometric Fluorescent Probe for Real-Time Detection of β-Galactosidase Activity in Lysosomes and Its Application in Drug-Induced Senescence Imaging.

Senescence is an important biological process, which leads to the gradual degradation of its physiological function and increases morbidity and mortality. Herein, a novel ratiometric fluorescent probe (P1) was constructed by using benzothiazolyl acetonitrile dye as fluorophore, exhibiting significantly enhanced blue-shifted emission to indicate the activity of β-galactosidase (β-gal), a commonly used biomarker for the detection of senescent cells. After incubation with β-gal, the excimer emission of P1 at 620 nm was weakened, while the emission at 533 nm was significantly enhanced, forming an obvious ratiometric probe with high sensitivity and low detection limit (2.7 mU·mL-1). More importantly, probe P1 can locate lysosomes accurately, allowing us to monitor the emergence of living cell senescence in real time. P1 was successfully used to detect β-gal activity in PC-12 cells, Hep G2 cells, and RAW 264.7 cells. It showed strong green fluorescence signal in senescent cells and red fluorescence signal in normal cells, indicating that it can detect endogenous senescence-related β-gal content in living cells. For in vivo drug-induced senescence imaging, after 5 weeks of injection of D-galactose or hydroxyurea, the mice showed significant fluorescence enhancement in specific channels to indicate the activity of β-gal in vivo. At the same time, the senescence of cell-specific organs and skin tissues at the organ level were also detected, which proved that the drug-induced senescence of brain, skin, and muscle tissues was the most serious. These results supported the important application value of P1 in senescence biomedical research.

A novel polymer-based probe for fluorescently ratiometric sensing of hydrogen sulfide with multiple applications

Hydrogen sulfide (H2S) as an endogenous signaling molecule, plays an irreplaceable role in many important physiological activities. It is also closely related to sewage treatment, wine quality evaluation, and food spoilage. Herein, we have successfully synthesized a novel polymer-based probe P1 for fluorescently ratiometric sensing of H2S with a high selectivity and sensitivity. By virtue of ring-opening metathesis polymerization (ROMP), P1 was prepared with the disulfide bond linked coumarin-norbornene dyad NB-SS-COU as energy donor, the aggregation-induced emission (AIE) fluorophore anchored norbornene NB-TPE as energy receptor, and the polyethylene glycol (PEG) attached norbornene NB-PEG as a hydrophilic chain. At the 400 nm excitation, P1 displays a bright red emission at 615 nm due to the efficient fluorescence resonance energy transfer (FRET) from energy donor COU to energy acceptor TPE. Upon addition of H2S, it shows strong COU-based blue emission at 473 nm for cleavage of the disulfide bond. We also constructed a smartphone sensing platform to conduct visual quantitative detection of H2S by calculating the B/R (blue/red) emission ratio values. Moreover, P1 can be successfully employed in evaluating the level fluctuations of endogenous and exogenous H2S in living cells, testing water samples/wine samples, and monitoring food freshness.

Ratiometric fluorescent probe with signals from two near-infrared channels for esterase activity and its application in biological imaging

Esterase serves as a crucial biomarker for the diagnosis of tumors due to its overexpression in cancer cells. Ratiometric fluorescent probe has the advantage of having two distinct emission wavelengths, which allows for the elimination of the effects of light intensity and optical path length by using the fluorescence intensity ratio between the two emission wavelengths. In this work, a novel near-infrared (NIR) fluorescent probe P1 for ratiometric monitor esterase activity was developed. Compared with ordinary ratiometric probes, both emission wavelengths of this probe are in the NIR region, which can effectively eliminate various fluctuations and blurriness in image contrast and achieve precise quantitative analysis. P1 itself shows a fluorescence emission at 717 nm. After incubated with esterase, a new emission peak at 822 nm emerged due to esterase's catalysis, and the fluorescence intensity at 717 nm steadily decreased, which achieved the highly sensitive detection of esterase with a detection limit calculated as 0.25 mU/mL. What’s more, P1 showed high selectivity for esterase and was successfully applied for biological imaging in living cells and in vivo.

Conversion to Trimolecular G-Quadruplex by Spontaneous Hoogsteen Pairing-Based Strand Displacement Reaction between Bimolecular G-Quadruplex and Double G-Rich Probes.

Bimolecular or tetramolecular G-quadruplexes (GQs) are predominantly self-assembled by the same sequence-identical G-rich oligonucleotides and usually remain inert to the strand displacement reaction (SDR) with other short G-rich invading fragments of DNA or RNA. Appealingly, in this study, we demonstrate that a parallel homomeric bimolecular GQ target of Tub10 d(CAGGGAGGGT) as the starting reactant, although completely folded in K+ solution and sufficiently stable (melting temperature of 57.7 °C), can still spontaneously accept strand invasion by a pair of short G-rich invading probes of P1 d(TGGGA) near room temperature. The final SDR product is a novel parallel heteromeric trimolecular GQ (tri-GQ) of Tub10/2P1 reassembled between one Tub10 strand and two P1 strands. Here we present, to the best of our knowledge, the first NMR solution structure of such a discrete heteromeric tri-GQ and unveil a unique mode of two probes vs one target in mutual recognition among G-rich canonical DNA oligomers. As a model system, the short invading probe P1 can spontaneously trap G-rich target Tub10 from a Watson-Crick duplex completely hybridized between Tub10 and its fully complementary strand d(ACCCTCCCTG). The Tub10 sequence of d(CAGGGAGGGT) is a fragment from the G-rich promoter region of the human β2-tubulin gene. Our findings provide new insights into the Hoogsteen pairing-based SDR between a GQ target and double invading probes of short G-rich DNA fragments and are expected to grant access to increasingly complex architectures in GQ-based DNA nanotechnology.

ClO– Induced Dual-Excitation Fluorescent Probes Responding to Diverse Testing Modes with Ratio Methodology

Single-excitation ratio fluorescent probes have enabled the output signal with high signal-to-noise ratio, but are still plagued with technique challenges, including signal distortion and limited application scenario. Herein, a dual-excitation near-infrared (NIR) fluorescent probe P1 of coumarin derivatives is constructed, showing high signal output ability in the visible region and high tissue penetration depth ability in the NIR region. As NIR probe P1 selectively recognizes ClO-, the emission signal in the visible region (480 nm) of P1 is enhanced during the recognition process. Meanwhile, the NIR emission (830 nm) of the conjugated system is weakened, finally realizing that ClO- triggered the dual-excitation (720/400 nm) ratio fluorescence signal detection and monitoring. The signal of detection in vitro has high responsiveness. Meanwhile, in the process of NIR monitoring in vivo, positive contrast imaging of fluorescence is constructed, which can accurately monitor ClO- changes over time. The current dual-excitation fluorescence-based data calibration and/or comparison method improves the application of the traditional single-excitation ratio fluorescence strategy and provide innovative detection tools for accurate measurement of fluorescence detection, with detection/monitoring modes suitable for different physiological environments.

Highly sensitive photoelectrochemical and electrochemical dual-mode biosensing of polynucleotide kinase based on multifunctional BiOBr0.8I0.2 /CuSCN composite and biocatalytic precipitation

Polynucleotide kinase (PNK) plays an important role in maintaining genome integrity and is directly related to many severe human diseases, so its detection is of great significance. To accurately and reliably detect PNK, a photoelectrochemical (PEC) - electrochemical (EC) dual-mode biosensor is created based on BiOBr0.8I0.2/CuSCN multifunctional nanocomposite. In this protocol, the nanocomposite loaded Au nanoparticles (AuNPs) are used to immobilize capture probe P1, while the signal probe P2 is labeled by glucose oxidase modified AuNPs (GOx@AuNPs-P2). They precisely hybridize to produce a double-stranded DNA (ds-DNA)-contained composite at the ITO electrode, which can be phosphorylated in the presence of PNK before being cleaved selectively by λ-exonuclease and leaving the electrode surface. The uncut ds-DNA with GOx@AuNPs-P2 catalyzes glucose to generate H2O2, and under the catalysis of peroxidase-like BiOBr0.8I0.2/CuSCN, 4-chlorohexadione precipitation is produced, which amplifies PEC and EC signals. Owing to the coordination effect of different elements, including enzyme and aptamers, the proposed PEC-EC dual-mode platform displays high sensitivity and selectivity. Furthermore, the sensor shows high repeatability and stability. Further experiments indicate it has excellent application potential in the detection and inhibitor screening of PNK.

Development of Two Efficient Dual‐Function Fluorescent Probes for Specific Recognition of Zn2+/H2S

AbstractIn this paper, two dual function fluorescent probes P1 and P2 with obvious fluorescence spectrum change were developed for the detection of Zn2+ and H2S. Density functional theory (DFT) was employed to investigate the detection mechanism. A further illustration of the fluorescence quenching mechanism was also studied by 1H NMR titration and UV‐Vis absorption spectroscopy. Both probes are new compounds containing coumarin structure as fluorophore. They can quantitatively detect Zn2+ and H2S in the concentration range from 0 to 50 μM with excellent selectivity and anti‐interference ability over many metal ions or anions. Most worthy is that probes P1 and P2 have better cell permeability with lower cytotoxicity, which can be used for fluorescence detection in MCF‐7 cells, as well as the detection of exogenous Zn2+ in organisms.

Ion acoustic waves in lunar wake plasma

Kinetic theory of low frequency electrostatic waves is carried out in the lunar wake plasma modelled by kappa electrons, kappa-beam electrons, Maxwell–Boltzmann distributed protons and doubly charged Helium ions. The present work is motivated by observation of electrostatic waves on the outbound side of the first lunar wake flyby of the mission Acceleration, Reconnection, Turbulence and Electrodynamics of the Moon’s Interaction with the Sun (ARTEMIS) probe P1. The dispersion characteristics of electrostatic waves which are identified as ion-acoustic waves are carried out for the observational plasma parameters. The frequencies of the ion acoustic waves derived from the model corresponding to peak growth rates are f⩽0.02fpe; (fpe being electron plasma frequency) which matches with the frequency of the waves observed by wave burst 1 (WB1) in the lunar wake (Tao et al., 2012). Our theoretical analysis reveals that in order to excite the low-frequency wave modes, low-energy electron beams are required which are not apparent in the observations.

Bacterial DNA analysis based on target aided self-assembly cycle amplification coupled with DNA-AgNCs/three-way DNA junction

We have constructed a simple, enzyme-free and label-free detection method for bacterial DNA. First, bacterial DNA (T) is designed as a trigger to make the designed metastable hairpin probes P1, P2, P3 (carrying DNA silver nanoclusters sequences) autonomously cross-open to achieve catalytic self-assembly, inducing a three-way DNA junction formation through the catalyzed hairpin self-assembly (CHA) mode. Then, G-rich sequences are introduced to form DNA silver nanoclusters G quadruplex (DNA-AgNCs-G quadruplex), and a strong fluorescence signal can be collected under the action of silver nitrate and sodium borohydride solutions. Through the fluorescent reporter of DNA silver nanoclusters (DNA-AgNCs) and the amplification of CHA, we successfully detected target DNA at concentrations as low as 19.95 fM. Compared with the constructed acyclic system, the sensitivity of this cyclic system is improved about three orders of magnitude. Moreover, this recycling system achieves good selectivity for mismatched target DNA and excellent recovery rate in practical samples. Therefore, bacterial DNA analysis based on target-aided self-assembly cycle amplification coupled with DNA-AgNCs/three-way DNA junction is successfully developed, promising its great application in biological sensing.

In-situ construction of fluorescent probes for hydrogen peroxide detection in mitochondria and lysosomes with on-demand modular assembling and double turn-on features

Due to the diverse H2O2 distribution in organelles, fluorescent probes were usually required to be prepared separately, which limited the convenience and practicability. Herein, we reported a flexible strategy to in-situ construct H2O2 fluorescent probes in different organelles. A tetrazine fused probe TP was developed with rapid click reaction capacity and sensitive H2O2 response. When treated with H2O2, the turn-on fluorescence was effectively quenched by the tetrazine part. Only after click reaction with dienophiles, the fluorescence resumed. In application, cells were firstly treated with triphenylphosphorus tagged norbornene (TPP-NB) to label mitochondria, which was followed by the introduction of probe TP to trigger click reaction. The in-situ constructed probe P1 served as a local H2O2 sensor. In a similar way, probe P2 was in-situ constructed in lysosomes via probe TP and morpholine tagged norbornene (MP-NB). With this on-demand modular assembling and double turn-on features, our strategy to construct fluorescent probes presented high flexibility and anti-interference performance, which was expected to inspired more applications in biological studies.

A dynamic thiol-disulfide exchange assisted high cell-penetrating fluorescent probe for intracellular H2S detection

Hydrogen sulfide (H2S) is a gaseous signaling transmitter which plays a crucial role in cell fates. Given the volatility of H2S in live cells corresponding to the various cell activities, it is important to design and synthesize novel fluorescent probes which could enter cells rapidly for the fast H2S detection. Herein, we reported a turn-on fluorescent probe P1 for H2S labeling in live cells, based on the dynamic thiol-disulfide exchange mechanism on cell surface, which facilitated the probe to enter cells quickly. High performance liquid chromatography (HPLC) results indicated the decoration of cell-penetrating disulfides endowed P1 with a high cellular uptake efficiency. Fluorescence microscopic imaging for HeLa cells suggested P1 was competent for intracellular H2S detection at submicromolar level. We expected our simple but effect strategy which enhanced the cell permeability of probes to benefit more accurate detection for intracellular specific molecule.

Rapid, Selective Fluorescent Determination of Copper (II) in Aqueous Solution and Living Cells Using a Dansyl-Based Click Probe

A novel 1,2,3-triazole based dansyl fluorophore has been designed and synthesized for detecting Cu2+ as a fluorescent probe (P1). The probe P1 exhibited high sensitivity and selectivity for Cu2+ compared to other selected species in aqueous solution within a wide pH span (5 to 14), which showed that P1 may be applied in the entire alkaline pH window. The limit of detection (LOD) was measured to be as low as 5 × 10−7 mol/L for Cu2+. A 1:2 binding ratio and association constant of 4.6 × 104 M−1 between P1 and Cu2+ were determined. Additionally, rapid response to Cu2+ was demonstrated in less than 3 seconds and the linear concentration range was from 0.5 to 2.5 µmol/L. Moreover, probe P1 showed good biocompatibility and cell membrane permeability and was successfully applied for imaging Cu2+ in water samples and living cells, which demonstrates the application of P1 in complicated environments.